Isolation and characterization of a cortical secretory vesicle membrane fraction.

1988 ◽  
Author(s):  
Carol A. Vater
Keyword(s):  
Membrane Fraction ◽  
Secretory Vesicle ◽  
Vesicle Membrane ◽  
Isolation And Characterization

scholarly journals Isolation and characterization of alkaline phosphatase-containing granules (phosphasomes) from human polymorphonuclear leucocytes

1985 ◽  
Vol 76 (1) ◽  
pp. 167-178
Author(s):  
G.P. Smith ◽  
G. Sharp ◽  
T.J. Peters
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Polymorphonuclear Leucocytes ◽  
Vesicle Membrane ◽  
Isolation And Characterization ◽  
Membrane Components ◽  
Counter Current

Human polymorphonuclear leucocytes were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation by centrifugation on a discontinuous sucrose density gradient to produce a phosphasome-enriched fraction, contaminated primarily by plasma membrane. Experiments to separate these membrane fractions by centrifugation on gradients of Nycodenz or Percoll, or by toroidal coil counter current chromatography, were unsuccessful. Fractionation carried out on neutrophils previously suspended in isotonic sucrose containing a low concentration of digitonin resulted in the preparation of a highly purified phosphasome fraction, free of plasma membrane components. Electron-microscope cytochemistry of the purified fraction identified the phosphasomes as regular and irregular-shaped spheres and rods, the alkaline phosphatase being associated with the inner aspect of the vesicle membrane. These granule structures were very similar to phosphasomes observed in intact neutrophils. A proportion of the endoplasmic reticulum and Golgi membrane marker enzymes remained associated with the phosphasomes throughout the separation procedures.

scholarly journals Characterization of intestinal microvillar membrane disks: detergent-resistant membrane sheets enriched in associated brush border myosin I (110K-calmodulin).

1989 ◽  
Vol 109 (3) ◽  
pp. 1153-1161 ◽  
Author(s):  
M S Mooseker ◽  
K A Conzelman ◽  
T R Coleman ◽  
J E Heuser ◽  
M P Sheetz
Keyword(s):  
Brush Border ◽  
Membrane Fraction ◽  
Average Rate ◽  
Isolation And Characterization ◽  
Myosin I ◽  
Latex Beads ◽  
Flat Disk ◽  
Nonionic Detergent ◽  
Actin Cables

The actin bundle within each microvillus of the intestinal brush border (BB) is tethered laterally to the membrane by bridges composed of BB myosin I. Avian BB myosin I, formerly termed 110K-calmodulin, consists of a heavy chain with an apparent Mr of 110 kD and three to four molecules of calmodulin "light chains." Recent studies have shown that this complex shares many properties with myosin including mechanochemical activity. In this report, the isolation and characterization of a membrane fraction enriched in bound BB myosin I is described. This membrane fraction, termed microvillar membrane disks, was purified from ATP extracts of nonionic detergent-treated microvilli prepared from avian intestinal BBs. Ultrastructural analysis revealed that these membranes are flat, disk-shaped sheets with protrusions which are identical in morphology to purified BB myosin I. The disks exhibit actin-activated Mg-ATPase activity and bind and cross-link actin filaments in an ATP-dependent fashion. The mechanochemical activity of the membrane disks was assessed using the Nitella bead movement assay (Sheetz, M. P., and J. A. Spudich. 1983. Nature [Lond.]. 303:31-35). These preparations were shown to be free of significant contamination by conventional BB myosin. Latex beads coated with microvillar membrane disks move in a myosin-like fashion along Nitella actin cables at rates of 12-60 nm/s (average rate of 33 nm/s); unlike purified BB myosin I, the movement of membrane disk-coated beads was most reproducibly observed in buffers containing low Ca2+.

scholarly journals Membrane retrieval in the guinea-pig neurohypophysis. Isolation and characterization of secretory vesicles and coated microvesicles after radiolabel incorporation in vivo

1984 ◽  
Vol 218 (2) ◽  
pp. 591-599 ◽  
Author(s):  
T Saermark ◽  
P M Jones ◽  
I C A F Robinson
Keyword(s):  
Guinea Pig ◽  
Biochemical Characterization ◽  
Hormone Secretion ◽  
Secretory Vesicle ◽  
Small Scale ◽  
Secretory Vesicles ◽  
Isolation And Characterization ◽  
Pituitary Glands

We have developed small-scale methods for the isolation and biochemical characterization of subcellular fractions from single guinea-pig posterior-pituitary glands. Secretory vesicles and coated microvesicles produced in this way were of similar purity to those isolated from large amounts of tissue by conventional ultracentrifugation. [35S]Cysteine injected into the hypothalamus was found in the soluble contents of secretory vesicles isolated from the neural lobes 24 h later. High-pressure liquid-chromatographic analysis revealed that the radiolabel was incorporated into the expected neurosecretory products (oxytocin, vasopressin and neurophysin) and also into a biosynthetic intermediate in the vasopressin system. The membranes of secretory vesicles were labelled with [3H]choline 24 h after its hypothalamic injection. Little or no [3H]choline could be demonstrated in coated microvesicles at this time, although these structures were labelled 5 days after injection. Stimulating hormone secretion by chronic dehydration produced a significant fall in [3H]choline content of the secretory-vesicle membranes without any transfer of label into coated microvesicles, suggesting that coated microvesicles are not involved in membrane retrieval in the neurohypophysis.

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