DEMONSTRATION OF A SPECIFIC CYTOSOL RECEPTOR IN THE NORMAL AND HYPERPLASTIC CANINE PROSTATE FOR 5α-ANDROSTANE-3α, 17α-DIOL

1975 ◽  
Vol 64 (3) ◽  
pp. 539-548 ◽  
Author(s):  
C. R. EVANS ◽  
C. G. PIERREPOINT
Keyword(s):  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Receptor Protein ◽  
Specific Receptor ◽  
Receptor Molecule ◽  
Sucrose Gradient Centrifugation ◽  
High Affinity

SUMMARY A specific receptor protein has been demonstrated for 5α-androstane-3α, 17α-diol in cytoplasmic extracts of normal and hyperplastic canine prostates. The receptor molecule, with a sedimentation coefficient of 4–5S, has been identified by the use of sucrose gradient centrifugation of tissue fractions which had been labelled in vitro with tritiated 5α-androstane-3α, 17α-diol. The receptor showed a relatively high affinity for this compound whereas binding could not be demonstrated with other labelled C19 steroids. In addition binding of tritiated 5α-androstane-3α, 17α-diol was not affected by the presence of 50-fold excesses of other C19 steroids.

scholarly journals Degradation of surfactant-associated protein B (SP-B) during in vitro conversion of large to small surfactant aggregates

1993 ◽  
Vol 295 (1) ◽  
pp. 141-147 ◽  
Author(s):  
R A W Veldhuizen ◽  
K Inchley ◽  
S A Hearn ◽  
J F Lewis ◽  
F Possmayer
Keyword(s):  
Surface Area ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Sucrose Gradient Centrifugation ◽  
Aggregate Fraction ◽  
Surfactant Aggregates ◽  
Morphological Studies ◽  
Surface Active ◽  
Protein B

Pulmonary surfactant obtained from lung lavages can be separated by differential centrifugation into two distinct subfractions known as large surfactant aggregates and small surfactant aggregates. The large-aggregate fraction is the precursor of the small-aggregate fraction. The ratio of the small non-surface-active to large surface-active surfactant aggregates increases after birth and in several types of lung injury. We have utilized an in vitro system, surface area cycling, to study the conversion of large into small aggregates. Small aggregates generated by surface area cycling were separated from large aggregates by centrifugation at 40,000 g for 15 min rather than by the normal sucrose gradient centrifugation. This new separation method was validated by morphological studies. Surface-tension-reducing activity of total surfactant extracts, as measured with a pulsating-bubble surfactometer, was impaired after surface area cycling. This impairment was related to the generation of small aggregates. Immunoblot analysis of large and small aggregates separated by sucrose gradient centrifugation revealed the presence of detectable amounts of surfactant-associated protein B (SP-B) in large aggregates but not in small aggregates. SP-A was detectable in both large and small aggregates. PAGE of cycled and non-cycled surfactant showed a reduction in SP-B after surface area cycling. We conclude that SP-B is degraded during the formation of small aggregates in vitro and that a change in surface area appears to be necessary for exposing SP-B to protease activity.

scholarly journals Biosynthesis of thyroglobulin. Incorporation of [1−14C]galactose, [1−14C]mannose and [4,5−3H2]leucine into soluble proteins by rat thyroids in vitro

1969 ◽  
Vol 112 (5) ◽  
pp. 709-719 ◽  
Author(s):  
Annette Herscovics
Keyword(s):  
Time Course ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Soluble Proteins ◽  
Protein Fractions ◽  
Sucrose Gradient Centrifugation ◽  
Carbohydrate Synthesis ◽  
Stepwise Manner ◽  
Rat Thyroid

1. Rat thyroid lobes were incubated for various periods of time in Krebs–Ringer bicarbonate containing [3H]leucine and either [1−14C]galactose or [1−14C]mannose. Radioactivity in soluble proteins was determined after their separation by sucrose-gradient centrifugation. 2. The time-course of incorporation of label from [14C]-mannose into soluble thyroid proteins was parallel to that observed for [3H]leucine. There was a lag of at least 30min. before either label appeared in non-iodinated thyroglobulin (protein 17–18s). During this time both labels were detected in two fractions known to contain subunit precursors of thyroglobulin (fractions 12s and 3–8s). Radioactivity from double-labelled fractions 12s and 3–8s was transferred to protein 17–18s during subsequent incubation in an unlabelled medium. 3. In contrast, most of the [14C]galactose was immediately incorporated into protein 17–18s. 4. During the first hour of incubation, puromycin almost completely inhibited the incorporation of label from [3H]leucine and [14C]mannose into all protein fractions, but had little effect on the incorporation of [14C]galactose into protein 17–18s. 5. These results indicate that mannose is incorporated into the carbohydrate groups of protein 17–18s at an earlier stage in its formation than galactose. It is suggested that the synthesis of the carbohydrate groups of ghyroglobulin begins soon after formation of the polypeptide components, more than 30min. before these are aggregated to protein 17–18s; carbohydrate synthesis then proceeds in a stepwise manner, galactose being incorporated at about the time of aggregation of subunits to protein 17–18s. Most, if not all, the carbohydrate is added to thyroglobulin before it is iodinated.

scholarly journals Hydrodynamic properties of adenosine Ri receptors solubilized from rat cerebral-cortical membranes

1987 ◽  
Vol 248 (3) ◽  
pp. 635-642 ◽  
Author(s):  
S M H Yeung ◽  
E Perez-Reyes ◽  
D M F Cooper
Keyword(s):  
Adenosine Receptor ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Guanine Nucleotides ◽  
Hydrodynamic Properties ◽  
High Affinity ◽  
Bivalent Cations ◽  
Cortical Membranes

Adenosine Ri receptors and inhibitory guanine-nucleotide-regulatory components were solubilized from rat cerebral-cortical membranes with sodium cholate. (-)-N6-Phenylisopropyl[2,8-3H]adenosine [(3H]PIA) binds with high affinity to the soluble receptors, which retain the pharmacological specificity of adenosine Ri receptors observed in membranes. The binding is regulated by bivalent cations and guanine nucleotides. Bivalent cations increase [3H]PIA binding by increasing both the affinity and the apparent number of receptors. Guanine nucleotides decrease agonist binding by increasing the dissociation of the ligand-receptor complex. Adenosine agonists stabilize the high-affinity form of the soluble receptor. The hydrodynamic properties of the adenosine receptor were determined with cholate extracts of membranes that were treated with [3H]PIA. Sucrose-gradient-centrifugation analysis indicates that the receptor has a sedimentation coefficient of 7.7 S. The receptor is eluted from Sepharose 6B columns with an apparent Stokes radius of 7.2 nm. Labelling of either sucrose-gradient or gel-filtration-column fractions with pertussis toxin and [32P]-NAD+ reveals that both the 41,000- and 39,000-Mr substrates overlap with the receptor activity. These studies suggest that the high-affinity adenosine-receptor-binding activity in the cholate extract represents a stable R1-N complex.

scholarly journals The construction and analysis of sucrose gradients for use with zonal rotors

1976 ◽  
Vol 159 (2) ◽  
pp. 259-265 ◽  
Author(s):  
W Hirst ◽  
R A Cox
Keyword(s):  
Iterative Procedure ◽  
Sucrose Gradient ◽  
Sucrose Solution ◽  
Sucrose Concentration ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Sucrose Gradient Centrifugation ◽  
Comparison With Experiment ◽  
Continuous Sucrose Gradient ◽  
Simple Equations

The rate of sedimentation of a particle in a sucrose solution depends on the viscosity and density of the medium. These two variables are related to the sucrose concentration and the temperature of the medium by new simple equations. These equations were used in a rapid iterative procedure that relates the distance moved by a zone in a continuous sucrose gradient to its sedimentation coefficient. It is shown by comparison with experiment that this iterative method allows the distance moved by a zone to be calculated rapidly. The method may therefore be used to optimize the separation of particles in a sucrose-gradient-centrifugation experiment. The method also allows the unknown sedimentation coefficients of several zones to be measured from a single sucrose-gradient-centrifugation experiment.

ANALYSIS OF RADIOIMMUNOASSAY BY DISC ELECTROPHORESIS AND SUCROSE GRADIENT CENTRIFUGATION

1971 ◽  
Vol 66 (2) ◽  
pp. 257-265 ◽  
Author(s):  
Lubomir Valenta ◽  
Michel L. Aubert
Keyword(s):  
Sucrose Gradient ◽  
Solid Phase ◽  
Gradient Centrifugation ◽  
Human Growth ◽  
Disc Electrophoresis ◽  
Separate Analysis ◽  
Adrenocorticotrophic Hormone ◽  
Sucrose Gradient Centrifugation ◽  
Before And After ◽  
Polypeptide Hormones

ABSTRACT Radioiodine-labelled synthetic adrenocorticotrophic hormone (ACTH), human growth hormone (HGH), human chorionic somato-mammotrophin (HCS), and human (HTSH) and bovine (BTSH) thyroid stimulating hormones were studied by disc-electrophoresis and sucrose gradient centrifugation before and after incubation with corresponding antisera. All antisera contained 7 S antibodies. After incubation, soluble antigenantibody complexes besides a small amount of precipitate were observed in the incubation mixture, characteristic of each hormone. The complexes migrated like gamma globulins or more slowly on disc-electrophoresis. and on sucrose gradient centrifugation showed patterns dependent on the time of incubation. Light 7 or 9 S, or < 12 S complexes occurred mostly after incubation for several minutes (up to 30 min) before analysis. When incubation was prolonged to 24 h and more, these relatively light complexes disappeared or diminished in favour of heavier soluble or precipitating complexes. Reproducibly obtainable sedimentation patterns of the soluble complexes suggested some definite recombination of antigen molecules with 7 S antibodies. The complexes did not occur on incubation with other sera than an antiserum to a given hormone. They were not influenced by EDTA. Displacement of the radioactivity of the complexes into the free hormone peak was obtained by addition of a non-labelled hormone identical with the labelled one. Sucrose gradient centrifugation and disc-electrophoresis are recommended for the study of immunoreaction of diluted materials and for a separate analysis of different steps of the radioimmunoassay. Radioimmunoassay was introduced for the measurement of protein hormones by Yalow & Berson (1960). The method, described originally for insulin, was later adapted to the detection of a number of protein and polypeptide hormones. On incubation of the hormone with its antiserum, a soluble antigenantibody complex is formed, which is separated from an excess of the free hormone by various methods, e. g. chromatoelectrophoresis, precipitation with a second antibody, adsorption on a solid phase etc. (Hunter 1967). Sucrose gradient centrifugation and disc-electrophoresis were occasionally used to follow some isolated aspect of radioimmunoassay (Fitschen 1965; Monjardino et al. 1968). We are demonstrating that these methods made it possible to analyze the radioimmunoassay step by step and thus may be useful for practical purposes as well as in a study of the immunoreaction of diluted materials.

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.

Purification and Characterization of a 3,17 β-Hydroxysteroid Dehydrogenase from Streptomyces hydrogenans

1979 ◽  
Vol 34 (7-8) ◽  
pp. 533-540 ◽  
Author(s):  
Helmut Duchmann ◽  
Lothar Träger
Keyword(s):  
Gel Electrophoresis ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Sedimentation Coefficient ◽  
Hydroxysteroid Dehydrogenase ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization

3,17 β-Hydroxysteroid dehydrogenase has been enriched and purified from cytosol of Streptomyces hydrogenans. After ammonium sulfate precipitation and filtration on Sephadex G-100 the enzyme was finally purified by preparative gel electrophoresis and DEAE-Sephadex A-50 chro­matography. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate gave a single band of mobility corresponding to molecular weight of 70 200 ± 2 500. 3 β-. 17 β- as well as 20 β-hydroxy steroids were dehydrogenated by the enzyme in the presence of NAD+. The dehydrogenation proceeded faster than the reduction of the corresponding ketosteroids in the presence of NADH. The enzyme does not accent NADP+ or NADPH as co-substrates. The apparent Km values were calculated to be 11 μᴍ for 5 α-dihydrotestosterone, 20 μᴍ for testosterone ana 68 μᴍ for epiandrosterone in the NAD+-driven reaction, 1.8 x 10-4 m for NADH+ and 1.9 x 10-4 ᴍ for NADH. The catalytic activity was influenced by the ratio of NAD+/ATP. The inhibition by ATP appears to be of a competitive type with respect to NAD+ (Ki 1.15 x 10-3 ᴍ).After sucrose gradient centrifugation in a preparative ultracentrifuge the enzyme sediments with 4.1 ± 0.1 S as estimated in comparison to other proteins of known sedimentation coefficient. The isoelectric point was determined to be 3.9 with the LKB preparative isoelectric focusing col­umn (pH 2-11) and 4.1 with the analytical flat bed polyacrylamide isofocusing (pH 3 - 5). The number of SH groups was determined to be 2 mol/mol enzyme. In the presence of 6 M urea the fig­ure inceases to 3 mol SH/mol enzyme. In the presence of an excess of p-chloromercuribenzoate the enzyme activity decreases only partially.

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