scholarly journals The secretion of tropoelastin by chick-embryo artery cells

1985 ◽  
Vol 230 (1) ◽  
pp. 217-225 ◽  
Author(s):  
N A Saunders ◽  
M E Grant
Keyword(s):  
Plasma Membrane ◽  
Chick Embryo ◽  
Microsomal Fraction ◽  
Single Gene ◽  
Subcellular Fractions ◽  
Secretory Vesicles ◽  
Half Time ◽  
Free System ◽  
Close Relationship ◽  
A Cell

Chymotryptic fingerprint analyses of tropoelastin a and tropoelastin b demonstrated a very close relationship between these two polypeptides synthesized in a cell-free system under the direction of chick-embryo polyribosomal mRNA. A similar study on tropoelastin polypeptides extracted in their hydroxylated and under-hydroxylated forms from artery cells incubated with [3H]valine in the absence and presence of alpha alpha'-bipyridine or 3,4-dehydroproline confirmed this close relationship and suggested that tropoelastins a and b are likely to be the products of a single gene. Pulse-chase experiments in which the synthesis and secretion of tropoelastin by artery cells were monitored demonstrated that, after a pulse with [3H]proline, the polypeptides rapidly appeared in the medium and the half-time of tropoelastin secretion was approx. 30 min. Further pulse-chase studies, in which [3H]tropoelastin contents of subcellular fractions were determined, showed that rough and smooth microsomal fractions contained maximal amounts of tropoelastin at different times. The quantity of tropoelastin in the smooth-microsomal fraction was always only a small proportion of that in the rough-microsomal fraction, suggesting rapid translocation of the polypeptides to the plasma membrane. Incubation of the cells with 0.1 mM-colchicine did not markedly alter the rate of secretion or the distribution of tropoelastin between the subcellular fractions, whereas when 1 microM-monensin was included in the incubations the polypeptides were retained in the rough microsomal fraction. The results are consistent with the proposal that tropoelastin may follow a pathway of secretion from rough endoplasmic reticulum to the plasma membrane via secretory vesicles.

scholarly journals In vitro reconstitution of exocytosis from plasma membrane and isolated secretory vesicles.

1985 ◽  
Vol 101 (6) ◽  
pp. 2263-2273 ◽  
Author(s):  
J H Crabb ◽  
R C Jackson
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Secretory Vesicles ◽  
Cytoplasmic Face ◽  
In Vitro Reconstitution ◽  
Transport Assay ◽  
Complex Preparation ◽  
A Cell ◽  
Vectorial Transport

We describe the reconstitution of exocytotic function through recombination of purified cortical secretory vesicles (CVs) and plasma membrane from sea urchin eggs. CVs were dislodged from a cell surface complex preparation by gentle homogenization in an isotonic dissociation buffer, and purified by differential centrifugation. CV-free plasma membrane fragments were obtained by mechanically dislodging CVs from cortical lawn (CL) preparations with a jet of CL isolation buffer. This procedure produced a "plasma membrane lawn" preparation, consisting of plasma membrane fragments attached via their vitelline layer (an extracellular glycocalyx) to a polylysine-coated microscope slide. When freshly prepared CVs were incubated with plasma membrane lawns, CVs reassociated with the cytoplasmic face of the plasma membrane, forming an exocytotically competent, reconstituted cortical lawn (RL). Exocytosis in RLs was monitored by phase-contrast microscopy, and quantitated with a sensitive microphotometric assay. Half-maximal exocytosis in RLs occurred at 18.5 microM free Ca2+; half-maximal exocytosis in control lawns occurred at 5.7 microM free Ca2+. Greater than 90% of the purified CVs that were not attached to a plasma membrane lawn remained intact when bathed in a buffer containing millimolar Ca2+. This result excluded the possibility that Ca2+-triggered CV lysis was responsible for our observations, and confirmed that the association of CVs with the plasma membrane was required for exocytosis in RLs. Evidence that the Ca2+-stimulated release of CV contents in CLs and RLs is the in vitro equivalent of exocytosis was obtained with an immunofluorescence-based vectorial transport assay, using an antiserum directed against a CV content protein: stimulation of RLs or partially CV-depleted CLs with Ca2+ resulted in fusion of the CV and plasma membranes, and the vectorial transport of CV contents from the cytoplasmic to the extracytoplasmic face of the egg plasma membrane.

Cinnamate-4-Hydroxyiase in Polyporus hispidus

1973 ◽  
Vol 51 (6) ◽  
pp. 731-734 ◽  
Author(s):  
C. P. Vance ◽  
A. M. D. Nambudiri ◽  
G. H. N. Towers
Keyword(s):  
Enzyme Activity ◽  
Enzymatic Activity ◽  
Microsomal Fraction ◽  
Mitochondrial Fraction ◽  
Maximum Activity ◽  
Coumaric Acid ◽  
Free System ◽  
Optimum Ph ◽  
A Cell ◽  
Plant Enzyme

A cell-free system capable of hydroxylating cinnamic acid to p-coumaric acid has been isolated from 10-day-oid cultures of Polyporus hispidus. The enzyme requires NADPH and FAD for maximum activity. Enzyme activity appears to be localized in the microsomal fraction with slight activity occurring in the mitochondrial fraction. The optimum pH for enzymatic activity is 7.5. This is the first report on any properties of this enzyme in a fungus. The enzyme differs from the known plant enzyme in its FAD requirement.

Gonadotropin-releasing hormone stimulates phospholipase C but not protein phosphorylation/dephosphorylation in plasma membrane from human epithelial ovarian cancer*

1993 ◽  
Vol 3 (5) ◽  
pp. 311-317 ◽  
Author(s):  
A. Imai ◽  
T. Ohno ◽  
T. Furui ◽  
K. Takahashi ◽  
T. Matsuda ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Plasma Membrane ◽  
Protein Phosphorylation ◽  
Inositol Phosphates ◽  
Specific Binding ◽  
Gonadotropin Releasing Hormone ◽  
Free System ◽  
Releasing Hormone ◽  
A Cell

In view of advances in treatment of certain hormone-dependent cancers with analogues of gonadotropin-releasing hormone (Gn-RH), this study was undertaken to establish the signal transduction events interacting with Gn-RH receptor in a cell-free system prepared from human ovarian mucinous cystadenocarcinoma samples. A high affinity specific binding (Kd=8 × 10−9 M) of [3H] Gn-RH was demonstrated in two from two plasma membrane preparations. Gn-RH showed no effects on the rate of protein phosphorylation from [γ-32P] adenosine triphosphate in the plasma membrane preparations. On the other hand, incubation of plasma membrane isolated form [3H]inositol-labeled specimens with Gn-RH in the presence of guanosine thiotriphosphate resulted in the rapid production of inositol phosphates. The Gn-RH effects was concentration-dependent, and half-maximal activation occurred with 1–3 nm Gn-RH. The Gn-RH-stimulated membrane event was observed in all plasma membrane isolations tested, but not in those from uterine endometrial carcinoma of a given case. These results provide the first direct evidence that Gn-RH receptor is coupled to phosphoinositide hydrolysis but not to certain membrane protein phosphorylation/dephosphorylation in ovarian carcinoma plasma membrane. Though the functional role of this event in human ovarian cancer is still obscure, it might be part of a possible point of attack for therapeutic approaches using Gn-RH analogues in this malignancy.

scholarly journals Docked Secretory Vesicles Undergo Ca2+-activated Exocytosis in a Cell-free System

1997 ◽  
Vol 272 (22) ◽  
pp. 14447-14453 ◽  
Author(s):  
Thomas F. J. Martin ◽  
Judith A. Kowalchyk
Keyword(s):  
Secretory Vesicles ◽  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Calcium Fluxes across the Plasma Membrane of Commelina communis L. Assayed in a Cell-Free System

1990 ◽  
Vol 93 (3) ◽  
pp. 940-947 ◽  
Author(s):  
Bettina Siebers ◽  
Peter Gräf ◽  
Elmar W. Weiler
Keyword(s):  
Plasma Membrane ◽  
Free System ◽  
Calcium Fluxes ◽  
Cell Free System ◽  
Commelina Communis ◽  
A Cell

scholarly journals Effects of cytochalasin B on membrane-associated microfilaments in a cell-free system.

1981 ◽  
Vol 91 (2) ◽  
pp. 524-530 ◽  
Author(s):  
M J Phillips ◽  
M Oda ◽  
I M Yousef ◽  
K Funatsu
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Mode Of Action ◽  
Actin Filaments ◽  
Cytochalasin B ◽  
Bile Canaliculus ◽  
Free System ◽  
A Cell

The mode of action of cytochalasin B was examined in vitro using bile canaliculus-enriched plasma membrane fractions isolated from rat liver. The pericanalicular microfilaments, which are mainly actin filaments and which are normally attached to the canalicular membranes, were dissociated from the membranes by cytochalasin B treatment. A microfilamentous network was found in the supernate of the cytochalasin B treatment. A microfilamentous network was found in the supernate of the cytochalasin-treated specimens and a number of polypeptides, of which a polypeptide corresponding in molecular weight to actin was a notable member. These results suggest that actin filaments become detached from the canaliculus membranes by cytochalasin B.

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