scholarly journals Vacuolar H+-ATPase of adrenal secretory granules. Rapid partial purification and reconstitution into proteoliposomes

1990 ◽  
Vol 271 (1) ◽  
pp. 127-131 ◽  
Author(s):  
J R Perez-Castiñeira ◽  
D K Apps
Keyword(s):  
Atp Hydrolysis ◽  
Secretory Granules ◽  
Gel Filtration ◽  
Partial Purification ◽  
Weak Base ◽  
Chromaffin Granule ◽  
Octyl Glucoside ◽  
Rapid Purification ◽  
Adrenal Chromaffin ◽  
Triton X

A procedure has been developed for the rapid purification and reconstitution into phospholipid vesicles of the proton-translocating ATPase of bovine adrenal chromaffin-granule membranes. It involves fractionation of the membranes with Triton X-114, resolubilization of the ATPase with n-octyl glucoside, addition of purified lipids and removal of detergent by gel filtration. The entire process can be completed within 2 h. H+ translocation was detected by the ATP-dependent quenching of the fluorescence of a permeant weak base. The effect of varying the lipid composition of the vesicles on ATP hydrolysis and H+ translocation by the reconstituted enzyme was examined. ATPase activity was maximally increased about 4-fold by added lipid, but was relatively insensitive to its composition, whereas vesicle acidification was absolutely dependent on the addition of phospholipids and cholesterol.

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

1990 ◽  
Vol 10 (2) ◽  
pp. 131-139
Author(s):  
Oyewole Adeyemo ◽  
E. O. Okegbile ◽  
O. O. Olorunsogo
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Boar Sperm ◽  
Sperm Membrane ◽  
Triton X

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.

Rat lingual lipase: partial purification, hydrolytic properties, and comparison with pancreatic lipase

1984 ◽  
Vol 247 (4) ◽  
pp. G385-G393 ◽  
Author(s):  
I. M. Roberts ◽  
R. K. Montgomery ◽  
M. C. Carey
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Pancreatic Lipase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Dodecyl Sulfate ◽  
Triton X

We have partially purified lingual lipase from the serous glands of rat tongue. With a combination of Triton X-100 extraction or Triton X-114 phase-separation techniques, Bio-Bead SM-2 treatment, dialysis, and gel filtration on Sephadex G-200 or Sephacryl S-300, we obtained a sparingly soluble lipid-free protein demonstrating hydrolytic activity against triglycerides and negligible phospholipase or cholesteryl esterase activities. Compared with homogenate, specific activities of the enzyme were enriched 3- to 5-fold prior to gel filtration and 10-fold after gel filtration. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions (6 M guanidine X HCl or 0.1% sodium dodecyl sulfate) revealed one major glycoprotein band with Mr approximately 50,000. Gel filtration of the active enzyme in 0.1% Triton X-100 gave an Mr approximately 270,000-300,000, suggesting extensive self-aggregation. With both tributyrin and triolein, the pH optimum of the purified enzyme was 4.0 and activity extended from pH 2.0 to 8.0. In contrast to purified human pancreatic lipase, lingual lipase hydrolyzed triglyceride emulsions and mixed micelles stabilized with both short-chain (dihexanoyl) and long-chain (egg) lecithin and were inhibited only slightly (18-25%) by micellar concentrations of two common bile salts, taurodeoxycholate and taurocholate. Our results suggest that the hydrolysis of dietary fat by lingual lipase may extend from the pharynx through the esophagus and stomach and into the upper small intestine.

Chromaffin cell cytoskeleton: its possible role in secretion

1985 ◽  
Vol 63 (6) ◽  
pp. 661-679 ◽  
Author(s):  
J.-M. Trifaró ◽  
M.-F. Bader ◽  
J.-P. Doucet
Keyword(s):  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Secretory Granules ◽  
Regulatory Proteins ◽  
Hormone Release ◽  
Chromaffin Granule ◽  
Calmodulin Antagonist ◽  
Calmodulin Binding ◽  
Cell Functions ◽  
Adrenal Chromaffin

Cytoskeleton proteins (actin, myosin, α-actinin, spectrin, tubulin, neurofilament subunits) and their regulatory proteins (calmodulin, gelsolin) have been isolated from adrenal chromaffin cells and characterized. Their physicochemical properties have been studied and their cell localizations have been revealed by biochemical, immunocytochemical, and ulstrastructural techniques. α-Actinin and spectrin are components of chromaffin granule membranes and some of the cell actin copurifies with these secretory granules. Myosin is not detected in the granules, but is present mainly in the cytosol and close to the cell surface. Trifluoperazine, a calmodulin antagonist, blocks stimulation-induced hormone release from chromaffin cells at a step distal from Ca2+ entry. High affinity calmodulin-binding sites have also been found in chromaffin granule membranes and their calmodulin-binding proteins have been characterized. Furthermore, microinjection of calmodulin antibodies into chromaffin cells blocks hormone output in response to stimulation. In view of the above findings, the possible roles of contractile proteins and calmodulin in chromaffin cell functions are discussed.

scholarly journals Nucleotide and bivalent cation specificity of the insulin-granule proton translocase

1983 ◽  
Vol 210 (1) ◽  
pp. 235-242 ◽  
Author(s):  
J C Hutton ◽  
M Peshavaria
Keyword(s):  
Atp Hydrolysis ◽  
Secretory Granules ◽  
Bivalent Cation ◽  
Nucleotide Hydrolysis ◽  
Storage Vesicles ◽  
Carbonyl Cyanide ◽  
Granule Membrane ◽  
Insulin Granule ◽  
Intracellular Storage ◽  
Adrenal Chromaffin

1. The nucleotide and bivalent cation specificity of the proton translocase activity of insulin secretory granules was investigated by assessing the inhibitor-sensitive rates of nucleotide hydrolysis by these organelles in relation to their chemiosmotic properties. 2. The relative rates of nucleotide hydrolysis by freeze/thawed granule preparations were: Mg2+ATP (100%) greater than Mg2+GTP (55%) greater than Mg2+UTP (48%) greater than Mg2+ITP (44%) greater than Mg2+CTP (23%) greater than Mg2+TTP (20%), and by intact granules were: Mg2+ATP (100%) greater than Mg2+ITP (74%) greater than Mg2+GTP (60%) greater than Mg2+CTP (35%). Mg2+ATP, Mg2+GTP and Mg2+ITP hydrolyses were inhibited by tributyltin and stimulated, in intact granules, by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Mg2+CTP hydrolysis was not markedly affected by these compounds. Correspondingly, only Mg2+ATP, Mg2+GTP and Mg2+ITP produced large changes in the delta psi and delta mu H+ across the granule membrane. 3. The relative rates of maximal ATPase activity stimulated by bivalent cations in freeze/thawed granule preparations were: Mg2+ (100%) greater than Mn2+ (82%) greater than Ca2+ (40%) greater than Co2+ (36%) greater than Zn2+ (0%), and in intact granules were: Mg2+ (100%) greater than Mn2+ (85%) greater than Co2+ (61%) greater than Ca2+ (42%). Tributyltin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone affected Mg2+-, Mn2+- and Co2+-activated, but not Ca2+-activated, ATP hydrolysis. Correspondingly, only Mg2+, Mn2+ and Co2+ supported the generation of a delta psi and delta mu H+ across granule membranes in the presence of ATP. 4. The results were consistent with a single proton translocase that had its catalytic site exposed on the external face of the granule membrane. The indicated specificity (Mg2+ATP = Mn2+ATP greater than Co2+ATP greater than Mg2+GTP greater than Mg2+ITP) was similar to that of enzymes described in membrane fractions prepared from adenohypophyseal tissue, adrenal chromaffin granules and yeast vacuoles. The insulin-granule activity thus appears to be a type of proton translocase, which is characteristic of intracellular storage vesicles in eukaryotic cells.

scholarly journals Partial purification and characterization of collagen galactosyltransferase from chick embryos

1976 ◽  
Vol 155 (1) ◽  
pp. 145-153 ◽  
Author(s):  
L Risteli ◽  
R Myllyä ◽  
K I Kivirikko
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Helical Conformation ◽  
Tissue Homogenate ◽  
Partial Purification ◽  
Molecular Weights ◽  
Soluble Collagen ◽  
Insoluble Collagen ◽  
Chick Embryo Extract ◽  
Triton X

Collagen galactosyltransferase was purified 50-150-fold from chick-embryo extract. The tissue homogenate was prepared in the presence of Triton X-100, since the addition of the detergent doubled the enzyme activity in the homogenate and the extract. Three species of the enzyme activity with different molecular weights were recovered on gel filtration, the mol.wts. being about 450000, 200000 and 50000. Collagen galactosyltransferase activity was strongly inhibited by p-mercuribenzoate, and stimulated by the addition of dithiothreitol to the incubation system. Studies on substrate requirements indicated that denatured citrate-soluble collagen is a more effective substrate than gelatinized insoluble collagen, as judged from their Km values. Experiments on three peptide fractions prepared from citrate-soluble collagen indicated that a fraction with an average mol.wt. of 500-600 contained peptides large enough to meet a minimun requirement for interaction with the enzyme. However, longer peptides were clearly better substrates. When native and heat-denatured citrate-soluble collagens were compared as substrates, practically no synthesis of galactosylhydroxylysine was found with native collagen. This finding suggests that the triple-helical conformation of collagen prevents the galactosylation of hydroxylysine residues.

scholarly journals Partial Purification and Characterisation of Alcohol Dehydrogenase from Acetobacter aceti Isolated from Palm Wine

2016 ◽  
Vol 8 (2) ◽  
pp. 232-236
Author(s):  
Donatus Chimaobi ONAH ◽  
Sabinus Oscar O. EZE ◽  
Patrick Emeka ABA
Keyword(s):  
Alcohol Dehydrogenase ◽  
Alcoholic Beverage ◽  
Gel Filtration ◽  
Effective Concentration ◽  
Partial Purification ◽  
Acetobacter Aceti ◽  
Palm Wine ◽  
Effects Of Ph ◽  
Polyethelene Glycol ◽  
Triton X

Palm wine is a very important alcoholic beverage whose consumption is limited because it spoils easily. The study was designed to isolate Acetobacter aceti from palm wine, then extract, purify and characterize alcohol dehydrogenase (AD) from the A. aceti. Muller Hilton agar was used as medium for the growth of A. aceti for 48 h. The cells were harvested and subjected to ultrasonication using 500 watt ultrasonicator. Enzyme assay was carried out in both the supernatant and pellet. The enzyme was precipitated by polyethelene glycol 6000 while gel filtration was used for purifying the enzyme. The effects of pH, temperature and substrate concentration on AD were evaluated. The isolated A. aceti was gram negative, rod shaped, catalase positive, oxidase negative and was able to oxidize acetic acid to CO2 and H2O. Triton X-100 (0.3%) was the most effective concentration in solubilizing the protein (AD), while 15% polyethelene glycol 6000 was the most effective concentration for the precipitation of AD. An optimal pH of 5 was obtained with an optimal temperature of 50 °C. The most appropriate to solubilize and precipitate AD were 0.3% triton X-100 and 15% polyethelene glycol 6000 respectively, while AD activity was reduced under acidic pH, as well as for low and high temperatures.

scholarly journals Glycosylation and transmembrane topography of bovine chromaffin granule p65

1991 ◽  
Vol 279 (3) ◽  
pp. 699-703 ◽  
Author(s):  
H B Tugal ◽  
F van Leeuwen ◽  
D K Apps ◽  
J Haywood ◽  
J H Phillips
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Synaptic Vesicles ◽  
Secretory Granules ◽  
Chromaffin Granules ◽  
Trypsin Treatment ◽  
Chromaffin Granule ◽  
Calmodulin Binding ◽  
N Terminus ◽  
Adrenal Chromaffin

The bovine homologue of p65, a calmodulin-binding protein located in the membranes of synaptic vesicles and endocrine secretory granules, has been studied by the use of monoclonal antibodies directed against this antigen and against dopamine beta-mono-oxygenase. The protein (apparent molecular mass 67 kDa; pI = 5.5-6.2) is partially degraded by treatment with neuraminidase or endoglycosidase F. Trypsin treatment of intact adrenal chromaffin granules or of granule membranes releases a soluble 39 kDa fragment of p65 which corresponds to the whole of its cytoplasmic domain. This domain contains both the epitope for the monoclonal antibody cgm67 and the calmodulin-binding site. The 20 amino acids at the N-terminus of this fragment are identical to part of the rat p65 sequence.

scholarly journals Lipolytic Potential ofAspergillus japonicusLAB01: Production, Partial Purification, and Characterisation of an Extracellular Lipase

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Lívia Tereza Andrade Souza ◽  
Jamil S. Oliveira ◽  
Vera L. dos Santos ◽  
Wiliam C. B. Regis ◽  
Marcelo M. Santoro ◽  
...  
Keyword(s):  
Room Temperature ◽  
Gel Filtration ◽  
Industrial Applications ◽  
Partial Purification ◽  
Extracellular Lipase ◽  
Inhibitory Effects ◽  
Ammonium Sulphate Precipitation ◽  
Lipase Enzyme ◽  
Alkaline Conditions ◽  
Triton X

Lipolytic potential ofAspergillus japonicusLAB01 was investigated by describing the catalytic properties and stability of a secreted extracellular lipase. Enzyme production was considered high under room temperature after 4 days using sunflower oil and a combination of casein with sodium nitrate. Lipase was partially purified by 3.9-fold, resulting in a 44.2% yield using ammonium sulphate precipitation (60%) quantified with Superose 12 HR gel filtration chromatography. The activity of the enzyme was maximised at pH 8.5, and the enzyme demonstrated stability under alkaline conditions. The optimum temperature was found to be 45°C, and the enzyme was stable for up to 100 minutes, with more than 80% of initial activity remaining after incubation at this temperature. Partially purified enzyme showed reasonable stability with triton X-100 and was activated in the presence of organic solvents (toluene, hexane, and methanol). Among the tested ions, only Cu2+, Ni2+, and Al3+showed inhibitory effects. Substrate specificity of the lipase was higher for C14 among various p-nitrophenyl esters assayed. TheKMandVmaxvalues of the purified enzyme for p-nitrophenyl palmitate were 0.13 mM and 12.58 umol/(L·min), respectively. These features render a novel biocatalyst for industrial applications.

scholarly journals A sodium/proton antiporter in chromaffin-granule membranes

1989 ◽  
Vol 257 (2) ◽  
pp. 499-507 ◽  
Author(s):  
J R Haigh ◽  
J H Phillips
Keyword(s):  
Atp Hydrolysis ◽  
Competitive Inhibitor ◽  
Alkaline Media ◽  
Secretory Vesicles ◽  
Chromaffin Granules ◽  
Weak Base ◽  
Chromaffin Granule ◽  
Fluorescence Measurements ◽  
Indirect Assay ◽  
Exchange Activity

Chromaffin granules, the secretory vesicles of the adrenal medulla, have a Na+/H+ exchange activity in their membranes which brings their proton gradient into equilibrium with a Na+ gradient. This explains why Na+ is mildly inhibitory to amine transport (which is driven by the H+ gradient) The activity can be demonstrated by using accumulation of 22Na+ in response to a pH gradient that is either imposed by diluting membrane ‘ghosts’ into alkaline media, or generated by ATP hydrolysis. It can also be monitored indirectly by fluorescence measurements in which the pH inside ‘ghost’ is monitored by quenching of a fluorescent weak base. This method has been used to monitor Na+ entry into acid-loaded Șghosts’ of H+ entry into methylamine accumulation. The exchanger appears to be reversible and non-electrogenic, with a stoichiometry of 1:1. Using an indirect assay we measured an apparent Km for Na+ of 4.7 mM, and a Ki for amiloride, a competitive inhibitor, of 0.26 mM. Direct assays using 22Na+ suggested a higher Km. Ethylisopropylamiloride was not inhibitory.

scholarly journals Extraction and Partial Purification of Mouse Uterine Alkaline Phosphatase

1979 ◽  
Vol 32 (2) ◽  
pp. 153 ◽  
Author(s):  
RN Murdoch ◽  
DJ Kay ◽  
WJ Capper
Keyword(s):  
Alkaline Phosphatase ◽  
Ammonium Sulfate ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Ammonium Sulfate Precipitation ◽  
Pregnant Mice ◽  
Triton X

Alkaline phosphatase in uterine homogenates from day 7 pregnant mice was solubilized using 0�2 % (v/v) Triton X-100 and extracted with 20% (v/v) n-butanol. The procedure, which resulted in 182- fold purification, included ammonium sulfate precipitation, DEAE-cellulose anion exchange chromatography and Sephadex 0200 gel filtration.

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