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.

Isolation of the 67Gallium Accumulating Fraction in Normal Rat Liver

1974 ◽  
Vol 13 (01) ◽  
pp. 72-84
Author(s):  
K. Hierholzer ◽  
K. zum Winkel ◽  
U. Haubold ◽  
E. Aulbert
Keyword(s):  
Electron Microscopy ◽  
Density Gradient ◽  
Intravenous Injection ◽  
Rat Liver ◽  
Soluble Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Differential Centrifugation ◽  
Normal Rat

SummarySubcellular 67Gallium distribution was investigated in normal rat liver after intravenous injection. By differential centrifugation and density gradient centrifugation 67Gallium accumulating bodies were isolated and identified as lysosomes by enzyme determination and electron microscopy. 67Gallium enrichment in this fraction was 23-fold. Using the isolated 67Gallium accumulating lysosomes the binding state of the isotope inside the lysosomes was studied. 67Gallium was found to be associated with the soluble fraction of lysosomes.

scholarly journals Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations

1994 ◽  
Vol 304 (2) ◽  
pp. 617-624 ◽  
Author(s):  
J C Osypiw ◽  
R L Allen ◽  
D Billington
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Rat Hepatocytes ◽  
Marker Enzymes ◽  
Cytotoxic Effects ◽  
Percoll Density Gradient Centrifugation ◽  
Cell Layers ◽  
Density Band ◽  
Percoll Density Gradient

Freshly isolated viable rat hepatocytes were separated into five subpopulations on shallow discontinuous Percoll density gradients. The periportal marker enzymes alanine aminotransferase (ALT), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) showed gradients of increasing activity from the subpopulation of least density (band 1, rho = 1.07 g/ml) to the subpopulation of greatest density (band 5, rho = 1.09 g/ml). The perivenous marker enzymes pyruvate kinase (PK) and glutamate dehydrogenase (GDH) showed gradients of decreasing activity from band-1 cells to band-5 cells. Glutamine synthetase (GS), which is confined to the two or three cell layers around the hepatic venule, was almost entirely restricted to band-1 hepatocytes. Band-5: band-1 ratios of enzyme activity were as follows: ALT, 8.0; LDH, 2.1; MDH, 1.6; GDH, 0.7; PK, 0.2; GS, 0.01. Band-5:band-1 ratios for ALT, LDH, PK and GS were maintained after culture of subpopulations in identical conditions for up to 72 h, whereas the ratios for MDH and GDH decreased and increased respectively towards unity. Band-1 hepatocytes exhibited greater cytotoxicity than band-5 cells after incubation with carbon tetrachloride or paracetamol. These perivenous-selective toxins produced greater decreases in cell viability and greater release of ALT and LDH from band-1 hepatocytes than from band-5 hepatocytes. Conversely, band-5 hepatocytes were more susceptible than band-1 hepatocytes to the cytotoxic effects of 1-naphthylisothiocyanate and methotrexate (known periportal-selective toxins). It is concluded that band-5 hepatocytes are enriched in periportal cells, whereas band-1 hepatocytes are enriched in perivenous cells. Isolation of hepatocyte subpopulations by Percoll density-gradient centrifugation has the considerable advantage that periportal and perivenous cells can be obtained from the same liver.

Acute hypertension provokes internalization of proximal tubule NHE3 without inhibition of transport activity

2002 ◽  
Vol 282 (4) ◽  
pp. F730-F740 ◽  
Author(s):  
Li Yang ◽  
Patrick K. K. Leong ◽  
Jennifer O. Chen ◽  
Nilem Patel ◽  
Sarah F. Hamm-Alvarez ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Transport Activity ◽  
Sprague Dawley ◽  
Acute Hypertension ◽  
Sprague Dawley Rats ◽  
Cortical Membranes ◽  
Apical Membranes

Acute hypertension rapidly decreases proximal tubule (PT) Na+ reabsorption, facilitated by a redistribution of PT Na+/H+exchangers (NHE3) out of the apical brush border, increasing NaCl at the macula densa, the signal for autoregulation of renal blood flow and GFR. This study aimed to determine whether NHE3 activity per transporter decreases during acute hypertension and the time dependence of the response. Blood pressure was elevated by 50–60 mmHg in male Sprague-Dawley rats for 5 or 30 min by constricting arteries. Renal cortical membranes were fractionated by density gradient centrifugation. NHE3 transport activity was assayed as the rate of appearance of acridine orange (AO) from AO-loaded vesicles in response to an inwardly directed Na+ gradient. After 5-min hypertension, 20% of total NHE3 protein, assayed by immunoblot, redistributed from low-density apical membranes to middensity membranes enriched in intermicrovillar cleft markers; by 30 min, a similar percentage shifted to heavier density membranes containing markers of endosomes. NHE3 activity shifted to higher density membranes along with NHE3 protein, that is, no change in activity/transporter during acute hypertension. Confocal analysis of NHE3 distribution also verified removal from apical microvilli and appearance in subapical vesicles. We conclude that the decrease in renal PT Na+ transport during acute hypertension is mediated by removal of transport-competent NHE3 from the apical brush border to subapical and internal reserves.

scholarly journals The subcellular loclization and properties of the ferrochelatase of etiolated barley

1976 ◽  
Vol 156 (2) ◽  
pp. 309-314 ◽  
Author(s):  
H N Little ◽  
O T G Jones
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Mitochondrial Fraction ◽  
Marker Enzymes ◽  
Mitochondrial Marker

The etioplast fraction prepared from dark-grown barley contained the enzyme ferrochelatase. A mitochondrial fraction prepared from the same dark-grown tissue also contained ferrochelatase. After density-gradient centrifugation an etioplast band was collected that was free from detectable mitochondrial marker enzymes and yet retained ferrochelatase activity. A membrane band that was enriched in mitochondria also contained ferrochelatase. The ferrochelatase in these two bands had different pH optima, but appeared very similar in their porphyrin specificity and their inhibition by metalloporphyrins.

scholarly journals Active Sugar Transport by the Small Intestine

1968 ◽  
Vol 52 (3) ◽  
pp. 482-494 ◽  
Author(s):  
Robert G. Faust ◽  
Mary G. Leadbetter ◽  
Regina K. Plenge ◽  
Alston J. McCaslin
Keyword(s):  
Small Intestine ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sugar Transport ◽  
Initial Step ◽  
Inhibitory Effect ◽  
Glucose Binding ◽  
Brush Borders

Tris-disrupted and intact brush border membrane preparations from mucosa of hamster jejunum were capable of preferentially binding actively transported D-glucose in a similar manner. Density gradient centrifugation of the Tris-disrupted brush borders indicated that D-glucose was bound to a fraction containing the cores or inner material of the microvilli. The properties of this binding were examined with the Tris-disrupted brush border preparation. Actively transported sugars competitively inhibited preferential D-glucose binding, whereas no effect was observed with nonactively transported sugars. Neither actively nor nonactively transported amino acids affected D-glucose binding. D-Glucosamine, which is not actively transported, was inhibitory to preferential D-glucose binding as well as to the active transport of D-glucose by everted sacs of hamster jejunum. No inhibitory effect was observed with the same concentration of D-galactosamine. Preferential D-glucose binding was also inhibited by sulfhydryl-reacting compounds, Ca2+, and Li+ ions. On the other hand, Mg2+ was shown to be stimulatory and Na+, NH4+, and K+ had no effect on this phenomenon. The results of these experiments suggest that preferential D-glucose binding to brush borders is related to the initial step in active sugar transport by the small intestine.

scholarly journals LYSOSOMES IN RAT THORACIC DUCT LYMPHOCYTES FRACTIONATED BY ZONAL CENTRIFUGATION

1973 ◽  
Vol 59 (1) ◽  
pp. 177-184 ◽  
Author(s):  
William E. Bowers
Keyword(s):  
Thoracic Duct ◽  
Light Microscope ◽  
Cathepsin D ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Differential Centrifugation ◽  
Acid Hydrolases ◽  
Fractionation Method

A method of zonal centrifugation was developed which separates rat thoracic duct lymphocytes (TDL) mainly according to size. The validity of the fractionation method was supported by light microscope observations, Coulter Counter sizing, and in vivo and in vitro labeling of lymphocytes. The distributions of lysosomal acid hydrolases in TDL fractionated by zonal centrifugation are similar to the distribution obtained for the cells. This result indicates that the large lymphocyte is not the sole bearer of either lysosomes or the large amount of soluble cathepsin D found in homogenates of TDL. Both reside mainly in small lymphocytes. This point was clearly established by fractionating homogenates of purified small lymphocytes by means of differential centrifugation and isopycnic density gradient centrifugation.

scholarly journals Incorporation of [14C]glucosamine and [14C]leucine into mouse kidney β-glucuronidase induced by gonadotrophin

1970 ◽  
Vol 117 (1) ◽  
pp. 161-167 ◽  
Author(s):  
Keitaro Kato ◽  
Hiroyuki Ide ◽  
Tsuranobu Shirahama ◽  
William H. Fishman
Keyword(s):  
Endoplasmic Reticulum ◽  
Microsomal Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Mouse Kidney ◽  
Differential Centrifugation ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
Glucuronidase Activity

Male BALB/C mice were injected intraperitoneally with 2.5 i.u. of gonadotrophin. After the injection, increase of β-glucuronidase activity was first observed in the microsomal fraction. By 36h 45–50% of the total homogenate activity was found in the microsomal fraction compared with 20–25% in the control microsomal fraction. From 36 to 80h not only microsomal β-glucuronidase but also lysosomal β-glucuronidase increased progressively. After 69h stimulation with 2.5 i.u. of gonadotrophin, d-[1-14C]glucosamine or l-[U-14C]leucine was injected intraperitoneally. After a further 3h the kidneys were homogenized and five particulate fractions were prepared by differential centrifugation. The β-glucuronidase in the microsomal and lysosomal fractions was released respectively by ultrasonication and by freezing and thawing treatment. The enzyme was purified by organic-solvent precipitation and by sucrose-density-gradient centrifugation. The results demonstrated the incorporation of these two labels into the mouse renal β-glucuronidase. The microsomal β-glucuronidase was much more radioactive than the lysosomal enzyme and approx. 80% of the newly synthesized enzyme appeared in microsomes and approx. 20% of that was found in lysosomes at this period. These results suggest that the mouse renal β-glucuronidase is a glycoprotein and that the newly synthesized enzyme is transported from endoplasmic reticulum to lysosomes.

Xanthacin. A bacteriocin of Myxococcus xanthus f b

1974 ◽  
Vol 20 (2) ◽  
pp. 131-135 ◽  
Author(s):  
Howard D. McCurdy Jr. ◽  
Thomas H. MacRae
Keyword(s):  
Molecular Sieve ◽  
Density Gradient Centrifugation ◽  
High Titer ◽  
Gradient Centrifugation ◽  
Myxococcus Xanthus ◽  
Sodium Dodecyl ◽  
Uranyl Acetate ◽  
Partial Purification ◽  
Differential Centrifugation ◽  
Varied Size

An extremely stable, particulate, bacteriocin, namely xanthacin, has been found in Myxococcus xanthus f b after mitomycin C induction. Xanthacin is resistant to trypsin, protease type VI, RNase, DNase, sodium dodecyl sulfate (SDS), acetone, ether, ultraviolet irradiation, and autoclaving. A high-titer preparation was obtained after partial purification by pervaporative concentration, differential centrifugation, molecular sieve chromatography, and density gradient centrifugation. Electron microscopy of platinum-shadowed and uranyl acetate stained preparations revealed the presence of circular bodies of varied size which resembled membrane fragments.

scholarly journals Intramitochondrial localization of δ-aminolaevulate synthetase and ferrochelatase in rat liver

1969 ◽  
Vol 114 (3) ◽  
pp. 455-461 ◽  
Author(s):  
Roxane McKay ◽  
R. Druyan ◽  
G. S. Getz ◽  
M. Rabinowitz
Keyword(s):  
Glutamate Dehydrogenase ◽  
Density Gradient ◽  
Malate Dehydrogenase ◽  
Rat Liver ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Membrane ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Differential Centrifugation ◽  
Inner Mitochondrial Membrane

Intramitochondrial loci for δ-aminolaevulate synthetase and ferrochelatase, the initial and final enzymes in haem synthesis, have been found in rat liver. Two different methods of fractionation were applied to mitochondria: (a) sonication and density-gradient centrifugation; (b) treatment with digitonin and differential centrifugation. Similar results were obtained with each technique. δ-Aminolaevulate synthetase is distributed similarly to two known matrix enzymes, malate dehydrogenase and glutamate dehydrogenase. Ferrochelatase is firmly bound to the the inner mitochondrial membrane. These results are considered in terms of the regulation of haem synthesis and in relation to mitochondrial biogenesis.

PSEUDOMONAS AERUGINOSA BACTERIOPHAGE SD1

1966 ◽  
Vol 12 (5) ◽  
pp. 885-893 ◽  
Author(s):  
P. D. Shargool ◽  
E. E. Townsend
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Thermal Denaturation ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Cesium Chloride ◽  
Defined Medium ◽  
Differential Centrifugation ◽  
Ordered Structure ◽  
Base Analysis ◽  
Denaturation Profile

A DNA-containing bacteriophage, designated SD1, was isolated from sewage, using strain B71 of Pseudomonas aeruginosa as host. Lysates titering 1 to 2 × 1011plaque-forming units/ml are produced by infecting cultures growing in a defined medium. Highly purified phage preparations were obtained by a procedure involving concentration with ammonium sulfate at pH 8.2, differential centrifugation, and density gradient centrifugation in cesium chloride solution.Electron microscopy revealed a structure possessing a head of regular hexagonal outline, 50 mμ in diameter, and a tail, 6.2 × 188 mμ. The phage contained approximately 2.8 × 10−17 g nitrogen, 8 × 10−18 g of phosphorus, and 1.1 × 10−16 g DNA per plaque-forming unit. Base analysis of SD1 DNA disclosed the presence of equimolar amounts of adenine and thymine and of guanine and cytosine; the latter two comprise 53% of the bases. The thermal denaturation profile of the isolated DNA indicates that SD1 DNA is a highly ordered structure: the guanine and cytosine content as estimated from the temperature of half maximal ultraviolet absorption agrees with that found by chemical analysis of the DNA.

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