Arginine esterase from isolated dog prostate secretory granules is fully active enzymatically

1985 ◽  
Vol 63 (12) ◽  
pp. 1603-1607 ◽  
Author(s):  
Gilles Frenette ◽  
Jean Y. Dubé ◽  
Jean R. Marcotte ◽  
Roland R. Tremblay
Keyword(s):  
Enzymatic Activity ◽  
Seminal Plasma ◽  
Density Gradient Centrifugation ◽  
Secretory Granules ◽  
Gradient Centrifugation ◽  
Protein Substrates ◽  
Inactive Form ◽  
Specific Enzymatic Activity ◽  
The One

We have isolated secretory granules from dog prostate homogenates and have determined whether a major portion of arginine esterase was localized in this fraction and if it was enzymatically active. Secretory granules were purified by density gradient centrifugation on sucrose, metrizamide, or Percoll. A major proportion of whole prostate homogenate arginine esterase was found in the granule fractions. Furthermore, the specific enzymatic activity in the granules was similar to the one observed in seminal plasma. No evidence could be found for the existence of significant amount of a zymogen inactive form of arginine esterase. These results suggest that arginine esterase could be active within the secretory granules in vivo and that it could hydrolyze protein substrates contained in this organelle.

FATE OF NEUROHYPOPHYSIAL GRANULE MEMBRANES AFTER SECRETION IN THE RAT: GENERATION OF A NEW APPARENT ORGANELLE BY FREEZING AND THAWING SECRETORY GRANULES

1981 ◽  
Vol 88 (1) ◽  
pp. 115-123
Author(s):  
JEAN H. LACEY ◽  
B. T. PICKERING
Keyword(s):  
Density Gradient Centrifugation ◽  
Secretory Granules ◽  
Gradient Centrifugation ◽  
Hormone Release ◽  
Freezing And Thawing ◽  
Granule Fraction ◽  
Two Peaks ◽  
Stimulation Of

The contents and membranes of the secretory granules in the rat neurohypophysis were labelled in vivo with [35S]cysteine and [3H]choline respectively. Density-gradient centrifugation of the labelled granules showed the membrane label to be distributed largely between two peaks: one associated with intact granules and one with the characteristics of empty granule envelopes. Stimulation of hormone release in vitro led to the movement of membrane label from the intact granule fraction to the other one, consistent with the recapture of membrane as large vesicles. Freezing and thawing the crude granule fraction, ostensibly to aid osmotic fracture, produced a single membrane component with a density intermediate between the two original fractions.

scholarly journals Oestradiol inhibits collagen breakdown in the involuting rat uterus

1972 ◽  
Vol 127 (4) ◽  
pp. 705-713 ◽  
Author(s):  
Janet N. Ryan ◽  
J. Frederick Woessner
Keyword(s):  
Cathepsin D ◽  
Density Gradient Centrifugation ◽  
Post Partum ◽  
Gradient Centrifugation ◽  
Specific Radioactivity ◽  
Rat Uterus ◽  
Oestradiol Treatment ◽  
Cathepsin D Activity ◽  
Stimulation Of

1. The earlier observation (Woessner, 1969) of oestradiol inhibition of collagen breakdown is confirmed and extended. Administration of 100μg of oestradiol-17β/day to parturient rats strongly inhibits the loss of collagen from the involuting uterus. Three experiments show that this effect is due to an inhibition of collagen degradation rather than to a stimulation of collagen synthesis. 2. Uterine collagen was labelled with hydroxy[14C]-proline by the administration of [14C]proline near the end of pregnancy. By 3 days post partum, control uteri lost 83% of their collagen and 90% of their hydroxy[14C]proline. Uteri from oestradiol-treated rats lost only 50% of both total and labelled hydroxyproline, with no decrease in the specific radioactivity of the hydroxyproline. 3. Incorporation of [14C]proline into uterine collagen hydroxyproline in vivo was not affected by oestradiol treatment. 4. Urinary excretion of hydroxyproline was increased in post-partum control rats and decreased in oestradiol-treated rats. 5. An enzyme capable of cleaving 4-phenylazobenzyloxycarbonyl-l-prolyl-l-leucylglycyl- l-prolyl-d-arginine (a substrate for clostridial collagenase) increased in activity in the post-partum uterus and was unaffected by oestradiol treatment. 6. Uterine homogenates digested uterine collagen extensively at pH3.2. This digestion was unaffected by the oestradiol treatment. 7. Lysosomal fractions prepared by density-gradient centrifugation of uterine homogenates contained coincident peaks of cathepsin D activity and peptide-bound hydroxyproline. The cathepsin D and hydroxyproline contents of this peak were unaffected by oestradiol treatment.

Density gradient centrifugation of sperm from a subfertile stallion and effect of seminal plasma addition on fertility

2011 ◽  
Vol 126 (1-2) ◽  
pp. 96-100 ◽  
Author(s):  
Gaetano Mari ◽  
Carolina Castagnetti ◽  
Giovanni Rizzato ◽  
Beatrice Mislei ◽  
Eleonora Iacono ◽  
...  
Keyword(s):  
Density Gradient ◽  
Seminal Plasma ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation

Subcellular localization of oxytocin in the ovine corpus luteum

1985 ◽  
Vol 63 (4) ◽  
pp. 309-314 ◽  
Author(s):  
G. E. Rice ◽  
G. D. Thorburn
Keyword(s):  
Subcellular Localization ◽  
Physicochemical Properties ◽  
Corpus Luteum ◽  
Density Gradient Centrifugation ◽  
Secretory Granules ◽  
Gradient Centrifugation ◽  
Particulate Fraction ◽  
Dense Granules ◽  
Oxytocin Release ◽  
Midluteal Phase

The subcellular localization of oxytocin within the corpus luteum of sheep was investigated using differential and density gradient centrifugation. Oxytocin was associated with a particulate fraction which sedimented to a density of 1.054 – 1.061 g/mL. The exclusion of [3H]oxytocin from this particulate fraction is indicative that particulate oxytocin represents endogenous compartmentalization. Particulate oxytocin, incubated in buffered medium at 37 °C, was stable for up to 1 h and the release of oxytocin was not affected by the pH of the incubation medium, over the range 5.5 – 8.5. Oxytocin release, however, was stimulated by incubating particle-bound oxytocin in buffered medium of low osmolality (<200 mosmol). These data are similar to the physicochemical properties reported for peptide-containing neurohypophysial secretory granules. Ultrastructural analysis of oxytocin-containing fractions revealed the presence of electron-dense granules (diameter, 200–250 nm). These data are suggestive that oxytocin, in the corpus luteum of sheep, is contained within a population of secretory granules which occur in high numbers during the midluteal phase of the oestrous cycle.

scholarly journals Separation of Degranulated Platelets from Normal Platelets

1977 ◽  
Author(s):  
P. Cieslar ◽  
J.P. Greenberg ◽  
M.A. Packham ◽  
R.L. Kinlough-Rathbone ◽  
J.F. Mustard
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Adenine Nucleotide ◽  
Thrombus Formation ◽  
Gradient Centrifugation ◽  
Rabbit Platelets ◽  
Fraction I

Platelets degranulated by thrombin (TDP) can be recovered, are effective in hemostasis and survive normally upon infusion into rabbits. Two approaches to determine whether platelets have been degranulated in vivo are: (1) measurement of circulating released materials; (2) detection of circulating degranulated platelets. We have used arabino-galactan (Stractan II) density gradient centrifugation to separate normal and degranulated platelets. The following distribution was obtained with washed rabbit platelets.The serotonin, PF4 and adenine nucleotide contents of the TDP were less than those of normal platelets and the TDP in fraction I had the lowest amounts. When TDP were labeled with 51cr and mixed with equal numbers of normal platelets, 85% of the platelets in fraction I were found to be TDP. 51Cr-TDP were injected into normal rabbits and reharvested after 18 hours. The greatest proportion of TDP was isolated in fraction I. Thus this method may make it possible to separate platelets that have lost their granule contents during participation in reversible thrombus formation in vivo.(* Visiting Fellow from the Faculty of Medicine, Charles University, Prague, Czechoslovakia.)

scholarly journals A study of nucleated erythrocytes by density-gradient centrifugation in a zonal rotor

1969 ◽  
Vol 111 (4) ◽  
pp. 583-591 ◽  
Author(s):  
A P Mathias ◽  
D. Ridge ◽  
N. St G. Trezona
Keyword(s):  
Molecular Weight ◽  
Ribosomal Rna ◽  
Base Composition ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Density Gradients ◽  
Avian Erythrocytes ◽  
Nuclear Rna ◽  
Zonal Rotor

1. Several substances of high molecular weight were examined for their suitability as suspension media in the formation of density gradients for the zonal centrifugation of avian erythrocytes. None proved satisfactory. 2. The behaviour of pigeon erythrocytes in rate-sedimentation experiments in a type A zonal rotor with density gradients of sucrose was examined. The mature cells sediment more rapidly than the younger cells and have a lower RNA/DNA ratio. Maturation is accompanied by a greater loss of RNA from the nucleus than from the cytoplasm. 3. The base composition of the nuclear RNA and of the two species of cytoplasmic ribosomal RNA is reported. 4. The RNA of erythrocytes may be labelled in vivo by injection of inorganic [32P]phosphate. The cells most active in the synthesis of RNA sediment less rapidly than the bulk of the cells. 5. Reticulocyte nuclei sediment more slowly than those from erythrocytes. Reticulocyte nuclei have a mean volume of 35μ3 and are isopycnic with sucrose of density 1·2871 (measured at 20°). Maturation of the nuclei causes them to shrink to a volume of 25μ3 and the density to increase to 1·2944.

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 Biosynthesis of respiratory-tract mucins. Incorporation of radioactive precursors into glycoproteins by canine tracheal explants in vitro

1973 ◽  
Vol 136 (4) ◽  
pp. 837-844 ◽  
Author(s):  
Daniel B. Ellis ◽  
Glenn H. Stahl
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sialic Acids ◽  
Equilibrium Density ◽  
Agarose Gels ◽  
Cscl Gradient ◽  
Tracheal Explants

1. Canine tracheal explants, cultured in medium 199, actively incorporated radioactive precursors into secreted macromolecules in vitro. 2. Puromycin, 6-diazo-5-oxo-l-norleucine and ouabain markedly inhibited the incorporation of these precursors. 3. Exogenous glucosamine at concentrations above 20mm caused a greater than 50% inhibition of the incorporation of l-[G-3H]fucose and l-[U-14C]serine. 4. Carbohydrate content of the purified secretions was approximately 50% and consisted principally of galactose, N-acetylglucosamine, N-acetylgalactosamine, fucose and sialic acids. 5. Chromatography on DEAE-cellulose and Bio-Gel A-150m and equilibrium density-gradient centrifugation in a CsCl gradient confirmed the presence of mucous glycoproteins. 6. Electrophoresis on 1% agarose gels gave profiles that were identical with canine respiratory mucus obtained in vivo. 7. These results support the utility of the explant system for studies of respiratory secretions.

scholarly journals The effect of thrombin on the density distribution of blood platelets: detection of activated platelets in the circulation

Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 433-438
Author(s):  
B van Oost ◽  
IH van Hien-Hagg ◽  
AP Timmermans ◽  
JJ Sixma
Keyword(s):  
Cardiopulmonary Bypass ◽  
Density Distribution ◽  
Density Gradient Centrifugation ◽  
Blood Platelets ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Human Platelets ◽  
Activated Platelets

The buoyant density of human platelets is decreased after they have been aggregated and induced to secrete their granule content by thrombin. This change in density was detected by discontinuous density gradient centrifugation using arabinogalactan (Stractan) solutions. The density decrease was dependent on the thrombin concentration and paralleled the extent of serotonin and beta-thromboglobulin secretion. The degranulated platelets maintained their integrity, and many of their functional properties. Mixtures of degranulated platelets and normal platelets could be resolved by Stractan gradient centrifugation and the number of degranulated platelets quantitated. Using this method, increased levels of less dense platelets were shown to occur after cardiopulmonary bypass. Assay of changes in platelet density by Stractan gradient centrifugation is a useful method for detection of activated platelets in vitro and in vivo.

Detection of antibodies to rabies virus by immunoelectron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 364-365
Author(s):  
R. K. Chaudhary ◽  
K. M. Charlton ◽  
M. T. Monette ◽  
A. E. Kelen
Keyword(s):  
Rabies Virus ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Standard Technique ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Live Virus ◽  
Human Diploid Cells ◽  
Diploid Cells

Immunization of humans and domesticated animals at risk of contracting rabies is common practice. The mouse neutralization test (MNT) is still the standard technique used for detecting and measuring antibody to rabies virus in sera of vacinees. However, it suffers from problems of reproducibility associated with tests performed in vivo. It also has the disadvantage of being expensive, time-consuming and hazardous. Hence, there has been a continuous search for a simple, rapid and safe test. In recent years, methods based on haemagglutination, haemadsorption, plaque reduction, immunofluorescence and radioimmunoassay have been developed, but none of them has eliminated the hazard involved in the use of live virus.With emphasis on laboratory safety, attempts were made to use inactivated rabies virus for antibody assay by immunoelectronmicroscopy (IEM), in comparison with the MNT.Inactivated rabies virus grown in human diploid cells was supplied by Connaught Laboratories Limited (CLL). The virus was purified by sucrose density gradient centrifugation and used at a concentration to give 35-50 particles per grid square.

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