Free-flow electrophoresis of the low-density structures that contain asialoglycoproteins in the rat liver

1984 ◽  
Vol 62 (11) ◽  
pp. 1051-1058 ◽  
Author(s):  
Maria T. Debanne ◽  
Maria Bolyos ◽  
Erwin Regoeczi
Keyword(s):  
Acid Phosphatase ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Complete Separation ◽  
Free Flow ◽  
Major Protein ◽  
Differential Centrifugation ◽  
Marker Enzymes ◽  
Minor Peak ◽  
A Minor

Rats were given an intravenous dose (1–2 μg/100 g) of iodine-labelled asialotransferrin, asialofetuin, or asialoorosomucoid either alone or in combinations, and the distribution of the radioactivity in the liver, removed 10–20 min after the injection, was analyzed by free-flow electrophoresis in an Elphor VaP 11 apparatus. Liver homogenates were prepared for electrophoresis according to an elaborate ultracentrifugation scheme that is outlined in detail with respect to conditions and yields. The scheme involved differential centrifugation, followed by density gradient centrifugation in a linear sucrose gradient and gel filtration using Sepharose 2B. Two ligand-containing fractions were obtained during differential centrifugation, each associated with a different complement of subcellular marker enzymes. On free-flow electrophoresis, the ligand present in either fraction exhibited a major and a minor peak. They were incompletely separated, the minor peak shouldering on the major one. The major peak had a higher electrophoretic mobility than the peaks of the acid phosphatase and phosphodiesterase I activities, but it had the same mobility as the sialyltransferase activity. The minor, less electronegative peak comigrated with the peaks of acid phosphatase and phosphodiesterase I activities and also with the major protein component of the subcellular fraction. It is concluded that the asialoglycoprotein-transporting subcellular vesicles are heterogeneous in regard to charge and that their complete separation from subcellular marker enzymes cannot be accomplished by free-flow electrophoresis.

scholarly journals LYSOSOMAL PHOSPHOLIPASES A1 AND A2 OF NORMAL AND BACILLUS CALMETTE GUERIN-INDUCED ALVEOLAR MACROPHAGES

1973 ◽  
Vol 56 (3) ◽  
pp. 621-627 ◽  
Author(s):  
Richard C. Franson ◽  
Moseley Waite
Keyword(s):  
Acid Phosphatase ◽  
Alveolar Macrophages ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Marker Enzymes ◽  
Sucrose Gradient Centrifugation ◽  
Control Distribution ◽  
Specific Activities ◽  
Selective Increase ◽  
Phospholipases A

A single intravenous injection of 0.1 mg of heat-killed Bacillus Calmette Guérin (BCG) in 0.1 ml of Bayol F produced an accumulation of activated alveolar macrophages (BCG induced). Cells were collected 3.5–4.0 wk after injection. Phospholipases A and three lysosomal marker enzymes (acid phosphatase, ß-glucuronidase, and lysozyme) were measured in homogenates, and the distribution of the phospholipases A and lysosomal, mitochondrial, and microsomal marker enzymes were examined after sucrose gradient centrifugation of a postnuclear (1,000 g) supernatant. Homogenates of normal and BCG-induced macrophages contained phospholipases A1 and A2 which had optimal activity at pH 4.0 in the presence of 2.0 mM ethylenediaminetetraacetate (EDTA). These activities were inhibited 50–70% by 2.0 mM CaCl2. Homogenates of BCG-induced macrophages had specific activities of ß-glucuronidase, acid phosphatase, and lysozyme, which were increased 1.5- to 3.0-fold over the controls, whether expressed as activity per mg protein or activity per 107 cells. The specific activities of the phospholipases A, on the other hand, were consistently lower than those of the control. Distribution of the phospholipases A and the lysosomal marker enzymes after sucrose gradient centrifugation suggested that the phospholipases A active at pH 4.0 in the presence of EDTA are of lysosomal origin since: (a) BCG treatment caused a selective increase in the density of particles which contained both the phospholipases A and three lysosomal marker enzymes; and (b) since the density of mitochondria and microsomes were not affected by BCG treatment. The increase in the density of lysosomes seen here may be related to previously described morphologic changes of BCG-induced alveolar macrophages.

Enhancing Effect of Surfactant and Protein on Hydrolysis of Thymolphthalein Monophosphate by Purified Prostatic Acid Phosphatase

1975 ◽  
Vol 21 (12) ◽  
pp. 1761-1765 ◽  
Author(s):  
Andras G Foti ◽  
Harvey Herschman ◽  
J Fenimore Cooper ◽  
Hedi imFeld
Keyword(s):  
Acid Phosphatase ◽  
Serum Proteins ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Prostatic Acid Phosphatase ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Brij 35 ◽  
Hydrolysis Of ◽  
Protein Bovine Serum Albumin

Abstract Purified prostatic acid phosphatase catalyzes the hydrolysis of thymolphthalein monophosphate 10-fold faster if an optimal concentration of Brij 35 (a wetting agent) or protein (bovine serum albumin or human serum proteins) is present. Results of gel filtration, dialysis, and sucrose density-gradient centrifugation analysis suggest that the substrate must combine with detergent or protein before the enzyme can catalyze its hydrolysis.

scholarly journals Use of glycyl-l-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles

1994 ◽  
Vol 300 (1) ◽  
pp. 229-236 ◽  
Author(s):  
T O Berg ◽  
P E Strømhaug ◽  
T Løvdal ◽  
P O Seglen ◽  
T Berg
Keyword(s):  
Acid Phosphatase ◽  
Degradation Products ◽  
Rat Hepatocytes ◽  
Endocytic Pathway ◽  
Marker Enzyme ◽  
Major Peak ◽  
Isolated Rat Hepatocytes ◽  
Cathepsin C ◽  
Minor Peak ◽  
A Minor

Lysosome-disrupting enzyme substrates have been used to distinguish between lysosomal and prelysosomal compartments along the endocytic pathway in isolated rat hepatocytes. The cells were incubated for various periods of time with 125I-labelled tyramine cellobiose (125I-TC) covalently coupled to asialoorosomucoid (AOM) (125I-TC-AOM); this molecule is internalized by receptor-mediated endocytosis and degraded in lysosomes, where the degradation products (acid-soluble, radio-labelled short peptides) accumulate, Glycyl-L-phenylalanine 2-naphthylamide (GPN) and methionine O-methyl ester (MOM), which are hydrolysed by lysosomal cathepsin C and a lysosomal esterase respectively, both diffused into hepatocytic lysosomes after electrodisruption of the cells. Intralysosomal accumulation of the hydrolysis products (amino acids) of these substrates caused osmotic lysis of more than 90% of the lysosomes, as measured by the release of acid-soluble radioactivity derived from 125I-TC-AOM degradation. The acid-soluble radioactivity coincided in sucrose-density gradients with a major peak of the lysosomal marker enzyme acid phosphatase at 1.18 g/ml; in addition a minor, presumably endosomal, acid phosphatase peak was observed around 1.14 g/ml. The major peak of acid phosphatase was almost completely released by GPN (and by MOM), while the minor peak seemed unaffected by GPN. Acid-insoluble radioactivity, presumably in endosomes, banded (after 1 h of 125I-TC-AOM uptake) as a major peak at 1.14 and a minor peak at 1.18 g/ml in sucrose gradients, and was not significantly released by GPN. GPN thus appears to be an excellent tool by which to distinguish between endosomes and lysosomes. MOM, on the other hand, released some radioactivity and acid phosphatase from endosomes as well as from lysosomes.

Subcellular Localization of Enterokinase in Human Duodenal Mucosa

1977 ◽  
Vol 53 (6) ◽  
pp. 551-562 ◽  
Author(s):  
R. W. Lobley ◽  
Ruth Franks ◽  
R. Holmes
Keyword(s):  
Brush Border ◽  
Soluble Fraction ◽  
Density Gradient Centrifugation ◽  
Sucrose Solution ◽  
Gradient Centrifugation ◽  
Total Activity ◽  
Duodenal Mucosa ◽  
Differential Centrifugation ◽  
Marker Enzymes ◽  
Suitable Marker

1. Specimens of human duodenal mucosa were obtained at duodenotomy. Superficial mucosal scrapings were homogenized in isotonic sucrose solution and fractionated by differential centrifugation. The distribution of organelles among the subcellular fractions was monitored by assay of suitable marker enzymes. 2. Enterokinase was recovered predominantly in the nuclear + brush-border fraction and 80% of the total activity was found to be particulate; approximately 20% of the enzyme was present in the soluble fraction, compared with 1% of the brush-border markers sucrase and alkaline phosphatase. 3. The brush-border-containing fraction was subfractionated by treatment with hypertonic Tris followed by differential and density gradient centrifugation. Enterokinase was distributed among the subfractions in parallel with brush-border markers and was concentrated in a subfraction which was highly enriched in microvillous membranes. 4. It was concluded that enterokinase is localized primarily to the microvillous membrane of the epithelial cell brush border in man, but that in addition a proportion of the enzyme may be present in a soluble or easily released form in the duodenal mucosa.

Characterization of immunoreactive dynorphin in human phaeochromocytomas

1988 ◽  
Vol 117 (1) ◽  
pp. 123-132 ◽  
Author(s):  
T. A. Howlett ◽  
G. M. Besser ◽  
L. H. Rees
Keyword(s):  
Acetic Acid ◽  
Gel Filtration ◽  
Molecular Size ◽  
Major Peak ◽  
Gel Filtration Chromatography ◽  
Post Translational Modifications ◽  
Minor Peak ◽  
Wet Weight ◽  
A Minor

ABSTRACT The prodynorphin-derived opioids, dynorphin (DYN) and α-neoendorphin (αNE) were studied in 24 human phaeochromocytomas and related tumours. Nineteen tumours, extracted in HCl (0·1 mol/l), contained concentrations of immunoreactive DYN (ir-DYN) ranging from < 0·5 to 794 pmol/g wet weight. None of the extracts in HCl contained ir-αNE (all < 2·4 pmol/g). Sephadex G-50 gel filtration chromatography of ir-DYN in HCl (0·1 mol/l) extracts of six tumours revealed three small peaks of ir-DYN of higher molecular size (approximately 12 000, 6000 and 3000 daltons), a minor peak of ir-DYN eluting just after DYN(1–17), and a broad major peak, consisting of at least three components, which was significantly retarded and eluted after the salt volume of the column. High-pressure liquid chromatography (HPLC) of these extracts revealed multiple peaks of ir-DYN, most of which did not coelute with any synthetic DYN peptides. On both gel filtration chromatography and HPLC, one of the minor peaks coeluted with DYN(1–32). None of the peaks of ir-DYN coeluted with DYN(1–17) which had been acetylated using acetic anhydride. Extracts of the same tumours in acetic acid (0·1 mol/l) yielded similar values for ir-DYN content, but parallelism in the assay was improved. Sephadex G-50 chromatography revealed a different pattern of ir-DYN with a major peak coeluting with DYN(1–17) and, in two tumours, a minor peak coeluting with DYN(1–8). Studies with HPLC revealed, however, that substantial degradation of synthetic DYN occurred during extraction in acetic acid (0·1 mol/l) in spite of the precautions taken. Phaeochromocytomas frequently contain ir-DYN in concentrations which may approach that of the mammalian pituitary. These tumours did not, however, contain ir-αNE and, with the possible exception of a small amount of DYN(1–32), the ir-DYN present did not correspond with any known sequences. Thus, whilst prodynorphin is expressed in phaeochromocytomas, it does not seem to be processed to the usual end-products, and post-translational modifications therefore seem likely. Enzymatic degradation of DYN may occur during extraction in acetic acid (0·1 mol/l), and this medium should, therefore, be avoided in studies of such labile peptides. J. Endocr. (1988) 117, 123–132

scholarly journals Fate of injected glucagon taken up by rat liver in vivo. Degradation of internalized ligand in the endosomal compartment

1990 ◽  
Vol 272 (3) ◽  
pp. 703-712 ◽  
Author(s):  
F Authier ◽  
M Janicot ◽  
F Lederer ◽  
B Desbuquois
Keyword(s):  
Acid Phosphatase ◽  
Plasma Membranes ◽  
Degradation Products ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Subcellular Fractionation ◽  
Chloroquine Treatment ◽  
Endosomal Acidification ◽  
Rapid Generation

The uptake and processing of glucagon into liver endosomes were studied in vivo by subcellular fractionation. After injection of [[125I]iodo-Tyr10]glucagon and [[125I]iodo-Tyr13]glucagon to rats, the uptake of radioactivity into the liver was maximum at 2 min (6% of the dose/g of tissue). On differential centrifugation, the radioactivity in the homogenate was recovered mainly in the nuclear (N), microsomal (P) and supernatant (S) fractions, with maxima at 5, 10 and 40 min, respectively; recovery of radioactivity in the mitochondrial-lysosomal (ML) fraction did not exceed 6% and was maximal at 20 min. On density-gradient centrifugation, the radioactivity associated first (2-10 min) with plasma membranes and then (10-40 min) with Golgi-endosomal (GE) fractions, with 2-5-fold and 20-150-fold enrichments respectively. Subfractionation of the GE fractions showed that, unlike the Golgi marker galactosyltransferase, the radioactivity was density-shifted by diaminobenzidine cytochemistry. Subfractionation of the ML fraction isolated at 40 min showed that more than half of the radioactivity was recovered at lower densities than the lysosomal marker acid phosphatase. Throughout the time of study, the [125I]iodoglucagon associated with the P, PM and GE fractions remained at least 80-90% trichloroacetic acid (TCA)-precipitable, whereas that associated with other fractions, especially the S fraction, became progressively TCA-soluble. On gel filtration and h.p.l.c., the small amount of degraded [125I]iodoglucagon associated with GE fractions was found to consist of monoiodotyrosine. Chloroquine treatment of [125I]iodoglucagon-injected rats caused a moderate but significant increase in the late recovery of radioactivity in the ML, P and GE fractions, but had little effect on the association of the ML radioactivity with acid-phosphatase-containing structures. Chloroquine treatment also led to a paradoxical decrease in the TCA-precipitability of the radioactivity associated with the P and GE fractions. Upon h.p.l.c. analysis of GE extracts of chloroquine-treated rats, at least four degradation products less hydrophobic than intact [125I]iodoglucagon were identified. Radio-sequence analysis of four of these products revealed three cleavages, affecting bonds Ser2-Gln3, Thr5-Phe6 and Phe6-Thr7. When GE fractions containing internalized [125I]iodoglucagon were incubated in iso-osmotic KCl at 30 degrees C, a rapid generation of TCA-soluble products was observed, with a maximum at pH 4. We conclude that endosomes are a major site at which internalized glucagon is degraded, endosomal acidification being required for optimum degradation.

Purification and Properties of Acid Phosphatase I From the Cellular Slime Mold Polysphondylium pallidum

1974 ◽  
Vol 52 (2) ◽  
pp. 126-136 ◽  
Author(s):  
Ilona A. Horgen ◽  
Paul A. Horgen ◽  
Danton H. O'Day
Keyword(s):  
Acid Phosphatase ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Neutral Hydrogen ◽  
Slime Mold ◽  
Cellular Slime Mold ◽  
Ph Optimum ◽  
Purification Method ◽  
Polysphondylium Pallidum ◽  
Cellular Slime

A procedure for the purification of a phosphomonoesterase, designated as acid phosphatase I, from the cellular slime mold Polysphondylium pallidum is described. Ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography are utilized in this purification method. The enzyme was judged to be homogeneous by gel filtration and by acylamide gel electrophoresis. The molecular weight of the enzyme was estimated by gel filtration and density gradient centrifugation to be 150 000 daltons. Acid phosphatase I was shown to be relatively heat stable, and it lost no activity when kept at 4 °C, pH 7.35, for over 30 days. The pH optimum was 3.5, but the enzyme was found to be more stable when kept near neutral hydrogen ion concentrations. P. pallidum acid phosphatase I was most effective using the natural substrates, fructose-1,6-pbosphate, β-glycerolphosphate, and 5′-mononucleotides. Various compounds including known phosphatase inhibitors were tested as to their effect on the activity of the enzyme. The slime-mold acid phosphatase appears in many ways to be a typical acid phosphomonoesterase.

Ultrastructural Characteristics of Platelets Separated from Plasma by ADP-Induced Aggregation

1976 ◽  
Vol 34 ◽  
pp. 164-165
Author(s):  
B.A. Shinoda ◽  
M.D. Hardison ◽  
S.F. Mohammad ◽  
H.Y.K. Chuang ◽  
R.G. Mason
Keyword(s):  
Blood Platelets ◽  
Gel Filtration ◽  
Differential Centrifugation ◽  
Ultrastructural Characteristics ◽  
Induced Aggregation

The utilization of blood platelets in experimentation frequently requires their separation from blood and subsequent resuspension in media of known composition. Several methods are available for preparation of isolated platelets (1-3) by differential centrifugation or gel filtration, but most methods are tedious and time consuming. Often platelets obtained by use of such methods are in a state different functionally and ultrastructurally from that of platelets in plasma (4).Recently Mohammad, Reddick, and Mason (5) reported a method in which platelets were separated from plasma by ADP-induced aggregation, washed several times, and then incubated in a carefully selected medium that resulted in deaggregation of platelets.

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