scholarly journals The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5′-nucleotidase

1986 ◽  
Vol 239 (1) ◽  
pp. 185-190 ◽  
Author(s):  
P Fritzson ◽  
T B Haugen ◽  
H Tjernshaugen
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Liver ◽  
Specific Activity ◽  
Ph Dependence ◽  
Gel Permeation Chromatography ◽  
Total Activity ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Distinct Fraction ◽  
Membrane Bound

An alkaline 5′-nucleotidase with properties similar to those of membrane-bound 5′-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.

Biosynthesis of 1,2-3H and 4-14C steroid sulfates of high specific activity

1970 ◽  
Vol 48 (1) ◽  
pp. 148-150 ◽  
Author(s):  
J. Torday ◽  
G. Hall ◽  
M. Schweitzer ◽  
C. J. P. Giroud
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Homogenate ◽  
High Specific Activity ◽  
Supernatant Fraction ◽  
Metabolic Studies

A supernatant fraction of rat liver homogenate enriched with ATP was used for the biosynthesis of the ester sulfates of several 3H and 14C steroids of the pregn-4-ene series. The method provides a simple means to prepare steroid sulfates of high specific activity for use in either metabolic studies or as reference compounds in the quantification of such conjugates by isotope assays.

scholarly journals Inactivation of glucose 6-phosphate dehydrogenase during germination and outgrowth of Bacillus cereus T endospores

1975 ◽  
Vol 148 (2) ◽  
pp. 259-268 ◽  
Author(s):  
M Orlowski ◽  
M Goldman
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Total Activity ◽  
Cellular Protein ◽  
Metabolic Energy ◽  
Supernatant Fraction ◽  
Stage Of Development ◽  
Glucose 6 Phosphate Dehydrogenase

The specific activity and total activity of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) under conditions of complete cell breakage fall 10-20-fold during a 3h period of spore germination and outgrowth. The spores must germinate (lose refractility), but do not have to undergo outgrowth, for the loss of activity to occur. Glucose 6-phosphate dehydrogenase activity from cells as any stage of development is completely stable in extracts at 4 degrees C or 30 degrees C. All of the enzyme activity is found in a soluble (50000g supernatant) fraction and remains completely soluble throughout development. Soluble protein and total cellular protein remain constant for about 2h. Proteinases could not be detected or protein turnover demonstrated during the morphogenetic process. Phenylmethanesuophony fluoride and o-phenanthroline, inhibitors of proteolytic enzymes, do not prevent glucose 6-phosphate dehydrogenase inactivation when added to whole cells. Mixing experiments show no inhibitor of glucose 6-phosphate dehydrogenase to be present in late-stage cells. The enzyme is not excreted into the culture medium. Chloramphenicol and rifampicine immediately stop protein synthesis and development but not the inactivation of glucose 6-phosphate dehydrogenase. NaN3, 2,4-dinitrophenol or anaerobiosis immediately stop development and prevent the loss of enzyme activity. A requirement for metabolic energy is therefore probable. Extracts of spores pre-labelled with L[14C]leucine were made at various stages of morphogenesis and subjected to polyacrylamide-gel electrophoresis. Glucose 6-phosphate dehydrogenase, which was identified by a specific stain, did not lose 14C label, and therefore may not be degraded during the inactivation process.

scholarly journals ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER

1971 ◽  
Vol 49 (3) ◽  
pp. 899-905 ◽  
Author(s):  
R. D. Cheetham ◽  
D. James Morré ◽  
Carol Pannek ◽  
Daniel S. Friend
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Specific Activity ◽  
Total Activity ◽  
Thiamine Pyrophosphate ◽  
Transferase Activity ◽  
Galactosyl Transferase ◽  
Thiamine Pyrophosphatase ◽  
Cell Components

The thiamine pyrophosphatase (the enzyme [s] catalyzing the release of inorganic phosphate with thiamine pyrophosphate as the substrate) activities of Golgi apparatus-, plasma membrane-, endoplasmic reticulum-, and mitochondria-rich fractions from rat liver were compared at pH 8. Activity was concentrated in the Golgi apparatus fractions, which, on a protein basis, had a specific activity six to eight times that of the total homogenates or purified endoplasmic reticulum fractions. However, only 1–3% of the total activity was recovered in the Golgi apparatus fractions under conditions where 30–50% of the UDPgalactose:N-acetylglucosamine-galactosyl transferase activity was recovered. Considering both recovery of galactosyl transferase and fraction purity, we estimate that approximately 10% of the total thiamine pyrophosphatase activity of the liver was localized within the Golgi apparatus, with a specific activity of about ten times that of the total homogenate. Cytochemically, reaction product was found in the cisternae of the endoplasmic reticulum as well as in the Golgi apparatus. This is in contrast to results obtained in most other tissues, where reaction product was restricted to the Golgi apparatus. Thus, enzymes of rat liver catalyzing the hydrolysis of thiamine pyrophosphate, although concentrated in the Golgi apparatus, are widely distributed among other cell components in this tissue.

Characterization studies on the membrane-bound adenosine triphosphatase (ATPase) of Azotobacter vinelandii

1975 ◽  
Vol 21 (11) ◽  
pp. 1807-1814 ◽  
Author(s):  
Peter Jurtshuk ◽  
John E. McEntire
Keyword(s):  
Electron Transport ◽  
Atpase Activity ◽  
Azotobacter Vinelandii ◽  
Specific Activity ◽  
Maximal Activity ◽  
Molar Ratio ◽  
Supernatant Fraction ◽  
Time Interval ◽  
Time Intervals ◽  
Membrane Bound

The adenosinetriphosphatase (ATPase) (EC 3.6.1.3) activity in Azotobacter vinelandii concentrates in the membranous R3 fraction that is directly associated with Azotobacter electron transport function. Sonically disrupted Azotobacter cells were examined for distribution of ATPase activity and the highest specific activity (and activity units) was consistently found in the particulate R3 membranous fraction which sediments on ultracentrifugation at 144 000 × g for 2 h. When the sonication time interval was increased, the membrane-bound ATPase activity could neither be solubilized nor released into the supernatant fraction. Optimal ATPase activity occurred at pH 8.0; Mg2+ ion when added to the assay was stimulatory. Maximal activity always occurred when the Mg2+:ATP stoichiometry was 1:1 on a molar ratio at the 5 mM concentration level. Sodium and potassium ions had no stimulatory effect. The reaction kinetics were linear for the time intervals studied (0–60 min). The membrane-bound ATPase in the R3 fraction was stimulated 12-fold by treatment with trypsin, and fractionation studies showed that trypsin treatment did not solubilize ATPase activity off the membranous R3 electron transport fraction. The ATPase was not cold labile and the temperature during the preparation of the R3 fraction had no effect on activity; overnight refrigeration at 4 °C, however, resulted in a 25% loss of activity as compared with a 14% loss when the R3 fraction was stored overnight at 25 °C. A marked inactivation (although variable, usually about 60%) did occur by overnight freezing (−20 °C), and subsequent sonication failed to restore ATPase activity. This indicates that membrane reaggregation (by freezing) was not responsible for ATPase inactivation. The addition of azide, ouabain, 2,4-dinitrophenol, or oligomycin to the assay system resulted in neither inhibition nor stimulation of the ATPase activity. The property of trypsin activation and that ATPase activity is highest in the R3 electron transport fraction suggests that its probable functional role is in coupling of electron transport to oxidative phosphorylation.

scholarly journals Stereospecificity of hepatic l-tryptophan 2,3-dioxygenase

1980 ◽  
Vol 189 (3) ◽  
pp. 393-405 ◽  
Author(s):  
Y Watanabe ◽  
M Fujiwara ◽  
R Yoshida ◽  
O Hayaishi
Keyword(s):  
Rat Liver ◽  
Mouse Liver ◽  
Specific Activity ◽  
Kinetic Studies ◽  
Assay Method ◽  
Supernatant Fraction ◽  
Subunit Structure ◽  
Apparent Homogeneity ◽  
Specific Activities ◽  
Assay Conditions

Tryptophan 2,3-dioxygenase [L-tryptophan–oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.11] has been reported to act solely on the L-isomer of tryptophan. However, by using a sensitive assay method with D- and L-[ring-2-14C]tryptophan and improved assay conditions, we were able to demonstrate that both the D- and L-stereoisomers of tryptophan were cleaved by the supernatant fraction (30000 g, 30 min) of liver homogenates of several species of mammals, including rat, mouse, rabbit and human. The ratio of activities toward D- and L-tryptophan was species variable, the highest (0.67) in ox liver and the lowest (0.07) in rat liver, the latter being hitherto exclusively used for the study of hepatic tryptophan 2,3-dioxygenase. In the supernatant fraction from mouse liver, the ratio was 0.23 but the specific activity with D-tryptophan was by far the highest of all the species tested. To identify the D-tryptophan cleaving enzyme activity, the enzyme was purified from mouse liver to apparent homogeneity. The specific activities toward D- and L-tryptophan showed a parallel rise with each purification step. The electrophoretically homogeneous protein had specific activities of 0.55 and 2.13 mumol/min per mg of protein at 25 degrees C toward D- and L-tryptophan, respectively. Additional evidence from heat treatment, inhibition and kinetic studies indicated that the same active site of a single enzyme was responsible for both activities. The molecular weight (150000), subunit structure (alpha 2 beta 2) and haem content (1.95 mol/mol) of the purified enzyme from mouse liver were similar to those of rat liver tryptophan 2,3-dioxygenase. The assay conditions employed in the previous studies on the stereospecificity of hepatic tryptophan 2,3-dioxygenase were apparently inadequate for determination of the D-tryptophan cleaving activity. Under the assay conditions in the present study, the purified enzyme from rat liver also acted on D-tryptophan, whereas the pseudomonad enzyme was strictly specific for the L-isomer.

scholarly journals Independent biosynthesis of soluble and membrane-bound alkaline phosphatases in the suckling rat ileum

1981 ◽  
Vol 200 (3) ◽  
pp. 645-654 ◽  
Author(s):  
Graeme P. Young ◽  
Steven T. Yedlin ◽  
David H. Alpers
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Double Diffusion ◽  
Soluble Form ◽  
Soluble Enzyme ◽  
Intestinal Alkaline Phosphatase ◽  
Absolute Increase ◽  
Membrane Bound

Enzymically active intestinal alkaline phosphatase exists in both soluble and membrane-bound forms in the suckling rat. Antiserum prepared against purified soluble alkaline phosphatase (anti-AlP) was shown to be monospecific when assessed by Ouchterlony double-diffusion analysis and immunoelectrophoresis. The two forms of alkaline phosphatase were antigenically identical and possessed similar affinities for anti-AlP. To study the biosynthesis of the two forms, 14-day-old rats were injected intraperitoneally with [3H]leucine. The labelling kinetics of alkaline phosphatase, extracted from supernatant and brush-border membrane fractions with anti-AlP, was followed over 20h. Incorporation of [3H]leucine into membrane-bound alkaline phosphatase was rapid, reaching a plateau at 6h. The soluble enzyme showed slower incorporation of label and maximal radioactivity was not reached until 12h after labelling, a lag of 6h behind the membrane-bound enzyme. Soluble alkaline phosphatase could not have been a precursor of the membrane form, as there was no early peak of radioactivity in the soluble form. To determine if the soluble enzyme was irreversibly derived from the membrane enzyme, a newly developed technique of labelling brush-border membrane proteins in vivo by intraluminal injection of diazotized [125I]iodosulphanilic acid was used. The appearance of 125I in soluble and membrane alkaline phosphatase was then monitored over a 7h period, encompassing the lag between maximal leucine labelling of the two forms. The results failed to show either a proportional transfer of radioactivity from membrane to soluble alkaline phosphatase or an absolute increase in radioactivity of the soluble form during degradation of brush-border alkaline phosphatase. Therefore there does not appear to be a serial precursor/product relationship between the soluble and membrane-bound forms of suckling-rat intestinal alkaline phosphatase.

Thyroid hormone binding in rat liver cytosol

1984 ◽  
Vol 103 (3) ◽  
pp. 265-271 ◽  
Author(s):  
G. Morgan
Keyword(s):  
Rat Liver ◽  
Binding Capacity ◽  
Supernatant Fraction ◽  
Gel Chromatography ◽  
Hormone Binding ◽  
Isolated Cells ◽  
Membrane Bound ◽  
Cell Cytosol ◽  
Better Than

ABSTRACT Binding of 3,5,3′-tri-iodothyronine (T3) and thyroxine (T4) to components of perfused rat liver supernatant fraction and isolated liver cell cytosol was studied. Of the four binding fractions in supernatant (X, A, Y and Z) separable by gel chromatography, both T3 and T4 bound preferentially to the A-fraction, which was shown to contain albumin as the major binding protein. When cytosol prepared from isolated cells was examined, T4 was again bound mainly in the A-fraction; however, T3 was observed to bind predominantly in the Y-region. Hormone binding to soluble protein in the latter system is thought to reflect the pattern in vivo, better than does binding in supernatant, although the possibility exists that the concentration of albumin observed in cytosol may be artifically high due to transfer of membrane-bound albumin during cell disruption. Nevertheless, albumin (possibly derived from more than one intracellular source) is capable of binding T4 in vivo. The presence of this protein within the hepatocyte may thus contribute to the high T4 binding capacity of the liver compared to other tissues. J. Endocr. (1984) 103, 265–271

scholarly journals Biosynthesis of microsomal nicotinamide–adenine dinucleotide phosphate–cytochrome c reductase by membrane-bound and free polysomes from rat liver

1969 ◽  
Vol 112 (2) ◽  
pp. 139-147 ◽  
Author(s):  
G. Ragnotti ◽  
G. R. Lawford ◽  
P. N. Campbell
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Specific Activity ◽  
Reductase Activity ◽  
Gel Filtration ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
High Specific Activity ◽  
Rat Liver Cells ◽  
Microsome Fraction ◽  
Membrane Bound

1. NADPH–ferricytochrome c oxidoreductase (EC 1.6.2.3) was purified from the endoplasmic reticulum of rat liver cells. The methods, which involved digestion of membrane with Steapsin, a crude pancreatic extract containing diastase and trypsin, gel filtration and preparative electrophoresis on polyacrylamide, provided an enzyme with a high specific activity in good yield. 2. The incorporation of 14C-labelled amino acids into the purified reductase by the incubation of various subcellular fractions was studied. The microsome fraction, bound polysomes, free polysomes and detergent-treated polysomes effected the synthesis of the enzyme. 3. The reductase that had been synthesized by the polysomes was tightly bound to preparations of smooth-surfaced endoplasmic reticulum that were added to the incubation medium. 4. Reductase activity could be detected on both free and detergent-treated polysomes. Evidence is presented to show that this activity was due, at least in part, to the presence on the ribosomes of nascent enzyme. The association of enzyme with detergent-treated polysomes did not appear to be due to contamination of the ribosomes with either membrane or cell sap but it is possible for such ribosomes to adsorb some enzyme. 5. The amount of reductase activity associated with the detergent-treated polysomes was increased when the rats from which the polysomes were derived had been previously injected with phenobarbitone. 6. The results are discussed with respect to their relevance for the question of the existence of two functionally different groups of polysomes in the liver and for current ideas on the biogenesis of membranes.

scholarly journals ENZYME-STRUCTURE RELATIONSHIPS IN THE ENDOPLASMIC RETICULUM OF RAT LIVER

1962 ◽  
Vol 15 (3) ◽  
pp. 541-562 ◽  
Author(s):  
Lars Ernster ◽  
Philip Siekevitz ◽  
George E. Palade
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Rat Liver ◽  
Membrane Fraction ◽  
Liver Microsomes ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Rat Liver Microsomes ◽  
Cytochrome C Reductase ◽  
Ribonucleoprotein Particles

Subfractionation of preparations of rat liver microsomes with a suitable concentration of sodium deoxycholate has resulted in the isolation of a membrane fraction consisting of smooth surfaced vesicles virtually free of ribonucleoprotein particles. The membrane fraction is rich in phospholipids, and contains the microsomal NADH-cytochrome c reductase, NADH diaphorase, glucose-6-phosphatase, and ATPase in a concentrated form. The NADPH-cytochrome c reductase, a NADPH (or pyridine nucleotide unspecific) diaphorase, and cytochrome b5 are recovered in the clear supernatant fraction. The ribonucleoprotein particles are devoid of, or relatively poor in, the enzyme activities mentioned. Those enzymes which are bound to the membranes vary in activity according to the structural state of the microsomes, whereas those which appear in the soluble fraction are stable. From these findings the conclusion is reached that certain enzymes of the endoplasmic reticulum are tightly bound to the membranes, whereas others either are loosely bound or are present in a soluble form within the lumina of the system. Some implications of these results as to the enzymic organization of the endoplasmic reticulum are discussed.

scholarly journals Detection of a Soluble Form of BACE-1 in Human Cerebrospinal Fluid by a Sensitive Activity Assay

2006 ◽  
Vol 52 (6) ◽  
pp. 1168-1174 ◽  
Author(s):  
Jan H Verheijen ◽  
Linda GM Huisman ◽  
Natascha van Lent ◽  
Ulf Neumann ◽  
Paolo Paganetti ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Specific Activity ◽  
Proteolytic Enzymes ◽  
Amyloid Β ◽  
Soluble Form ◽  
Amyloid Β Peptide ◽  
Activity Assay ◽  
Human Cerebrospinal Fluid ◽  
Membrane Bound ◽  
Bace 1

Abstract Background: Formation of deposits of the insoluble amyloid β-peptide is believed to be causally related with neurodegeneration in Alzheimer disease (AD). The β-peptide originates from a larger amyloid precursor protein (APP) by the action of proteolytic enzymes. The first proteolytic event leading to amyloid formation is the cleavage of APP by the membrane-bound aspartyl protease BACE-1, also known as memapsin-2. Inhibition of BACE-1 is thought to be a therapeutic approach to AD. Measuring BACE-1 activity in biological samples would be useful to elucidate the mechanism of AD and for development of AD drugs. Methods: We developed a sensitive and specific activity assay for BACE-1. The assay is based on a genetically engineered proenzyme that is specifically activated by BACE-1. The resulting active enzyme is measured with a chromogenic substrate. The use of 2 coupled reactions produces a detection limit as low as 0.4 pmol/L. Results: The assay detected BACE-1 activity in extracts of human brain tissue as well as, unexpectedly, in human cerebrospinal fluid (CSF). Gel electrophoresis and Western blotting identified the BACE-1 present in CSF as a truncated soluble form of the originally membrane-bound BACE-1. Conclusion: Detection of the soluble form of BACE-1 in CSF, a relatively easily accessible biological fluid, may be useful for monitoring the effects of drug candidates in vivo and may have diagnostic or prognostic applications.

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