Trypsin-uncoupled synthesis and secretion of yeast invertase: implications for the mechanism of secretion

1979 ◽  
Vol 25 (4) ◽  
pp. 528-534 ◽  
Author(s):  
Bruce E. Holbein ◽  
Denis K. Kidby
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
External Surface ◽  
Internal Surface ◽  
Low Molecular Weight ◽  
Synthetase Activity ◽  
Trypsin Treatment ◽  
Yeast Invertase

The subcellular distribution of invertase was examined after synthesis and secretion by sphaeroplasts had been uncoupled by the addition of 30 μg mL−1 trypsin. Sphaeroplasts secreted only the high molecular weight invertase during uncoupling by trypsin. The level of low molecular weight, 'small' invertase in the soluble internal pool was found to be elevated by over fivefold, and the membrane-associated pool was found to contain low molecular weight invertase in addition to intermediate molecular weight invertase, after 1.5 h of trypsin treatment. Purified plasma membranes from trypsin-treated sphaeroplasts had no detectable mannan synthetase activity. On the basis of these and previous findings, a working hypothesis wherein invertase is synthesized on the internal surface of the plasma membrane and glycosylated during its transit to the external surface is presented.

A modified procedure for studying enzyme secretion in yeast sphaeroplasts: subcellular distribution of invertase

1976 ◽  
Vol 22 (7) ◽  
pp. 989-995 ◽  
Author(s):  
Bruce E. Holbein ◽  
Cecil W. Forsberg ◽  
Denis K. Kidby
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
High Molecular Weight ◽  
Subcellular Distribution ◽  
Invertase Activity ◽  
Enzyme Secretion ◽  
Low Molecular Weight ◽  
Triton X

A significantly modified procedure for investigating enzyme secretion from yeast sphaeroplasts, and results from its application are described. Sphaeroplasts were derepressed for invertase biosynthesis in the presence of helicase and fractionated to reveal the distribution of high and low molecular weight forms of invertase. Secreted enzyme was found to be of high molecular weight, exclusively. Less than 10% of the total invertase activity was present in washed sphaeroplasts and of this, 43% was soluble, consisting of both high and low molecular weight forms of invertase. Washed membranes retained 32% of the internal invertase activity, and on solubilization with Triton X-100 the enzyme was found to be of an intermediate molecular weight. These results are consistent with the hypothesis that invertase is glycosylated at the plasma membrane.

P2Z purinoceptor-associated pores induced by extracellular ATP in macrophages and J774 cells

1997 ◽  
Vol 273 (6) ◽  
pp. C1793-C1800 ◽  
Author(s):  
Robson Coutinho-Silva ◽  
Pedro Muanis Persechini
Keyword(s):  
Molecular Weight ◽  
Bone Marrow ◽  
Patch Clamp ◽  
Plasma Membranes ◽  
Extracellular Atp ◽  
Low Molecular Weight ◽  
Whole Cell ◽  
Voltage Dependent ◽  
J774 Cells ◽  
Bone Marrow Derived Cells

Millimolar concentrations of extracellular ATP (ATPo) can induce the permeabilization of plasma membranes of macrophages and other bone marrow-derived cells to low-molecular-weight solutes, a phenomenon that is the hallmark of P2Z purinoceptors. However, patch-clamp and whole cell electrophysiological experiments have so far failed to demonstrate the existence of any ATPo-induced P2Z-associated pores underlying this permeabilization phenomenon. Here, we describe ATPo-induced pores of 409 ± 33 pS recorded using cell-attached patch-clamp experiments performed in macrophages and J774 cells. These pores are voltage dependent and display several properties of the P2Z-associated permeabilization phenomenon: they are permeable to both large cations and anions, such as tris(hydroxymethyl)aminomethane, N-methyl-d-glucamine, and glutamate; their opening is favored at temperatures higher than 30°C; they are blocked by oxidized ATP and Mg2+; and they can be triggered by 3′- O-(4-benzoylbenzoyl)-ATP but not by UTP or ADP. We conclude that the pores described in this report are associated with the P2Z permeabilization phenomenon.

scholarly journals Murine fetal liver macrophages bind developing erythroblasts by a divalent cation-dependent hemagglutinin.

1988 ◽  
Vol 106 (3) ◽  
pp. 649-656 ◽  
Author(s):  
L Morris ◽  
P R Crocker ◽  
S Gordon
Keyword(s):  
Plasma Membrane ◽  
Divalent Cation ◽  
Plasma Membranes ◽  
Fetal Liver ◽  
Divalent Cations ◽  
Membrane Receptors ◽  
Erythroid Cells ◽  
Mammalian Development ◽  
Trypsin Treatment ◽  
Plasma Membrane Receptors

During mammalian development the fetal liver plays an important role in hematopoiesis. Studies with the macrophage (M phi)-specific mAb F4/80 have revealed an extensive network of M phi plasma membranes interspersed between developing erythroid cells in fetal liver. To investigate the interactions between erythroid cells and stromal M phi, we isolated hematopoietic cell clusters from embryonic day-14 murine fetal liver by collagenase digestion and adherence. Clusters of erythroid cells adhered to glass mainly via M phi, 94% of which bound 19 +/- 11 erythroblasts (Eb) per cell. Bound Eb proliferated vigorously on the surface of fetal liver M phi, with little evidence of ingestion. The M phi could be stripped of their associated Eb and the clusters then reconstituted by incubation with Eb in the presence of divalent cations. The interaction required less Ca++ than Mg++, 100 vs. 250 microM for half-maximal binding, and was mediated by a trypsin-sensitive hemagglutinin on the M phi surface. After trypsin treatment fetal liver M phi recovered the ability to bind Eb and this process could be selectively inhibited by cycloheximide. Inhibition tests showed that the Eb receptor differs from known M phi plasma membrane receptors and fetal liver M phi did not bind sheep erythrocytes, a ligand for a distinct M phi hemagglutinin. We propose that fetal liver M phi interact with developing erythroid cells by a novel nonphagocytic surface hemagglutinin which is specific for a ligand found on Eb and not on mature red cells.

Ca2+-sensitive permeability changes caused by influenza virus

1983 ◽  
Vol 3 (8) ◽  
pp. 749-755 ◽  
Author(s):  
Kalpana Patel ◽  
Charles A. Pasternak
Keyword(s):  
Molecular Weight ◽  
Influenza Virus ◽  
Plasma Membrane ◽  
Trypan Blue ◽  
Low Molecular Weight ◽  
Permeability Change ◽  
Permeability Changes ◽  
Phosphorylated Compounds

Influenza virus added to Lettré cells at pH 5.3 induces a permeability change similar to that elicited by Sendal virus at pH 7.4: K+ and Na+ equilibrate across the plasma membrane and low-molecular-weight phosphorylated compounds leak out of cells, which remain impermeable to trypan blue.

scholarly journals Oxygen metabolism of mammalian spermatozoa. Generation of hydrogen peroxide by rabbit epididymal spermatozoa

1981 ◽  
Vol 198 (2) ◽  
pp. 273-280 ◽  
Author(s):  
Michael K. Holland ◽  
Bayard T. Storey
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Cell Concentration ◽  
Oxygen Metabolism ◽  
Low Molecular Weight ◽  
H2o2 Production ◽  
Epididymal Spermatozoa ◽  
Cell Concentrations ◽  
Osmotic Treatment ◽  
Rabbit Spermatozoa

Rabbit spermatozoa from the cauda epididymis produced 0.7–0.8nmol of H2O2/min per 108 cells at cell concentrations below 107 cells/ml with linear dependence on cell concentration. Above 2 × 107 cells/ml, the rate again became linear with cell concentration but decreased to 0.1–0.2nmol/min per 108 cells. Spermatozoa treated with amphotericin B, which makes the plasma membrane highly permeable to low-molecular-weight compounds, showed a similar dependence of H2O2 production rate on cell concentration; below 107 cells/ml the rate was 0.3–0.4nmol/min per 108 cells; above 2 × 107 cells/ml, the rate was 0.1–0.2nmol/min per 108 cells. Hypo-osmotically treated rabbit epididymal spermatozoa, a preparation useful for studying mitochondrial function in sperm [Keyhani & Storey (1973) Biochim. Biophys. Acta305, 557–565] produced 0.1–0.2nmol/min per 108 cells in the absence of added substrates. The dependence of rate on cell concentration was linear from 107 to 2.2 × 108 cells/ml. This endogenous rate was unaffected by rotenone, but stimulated 4-fold by antimycin A. Addition of the mitochondrial substrates lactate plus malate increased the rate of H2O2 production to 0.3nmol/min per 108 cells. The decreased rate of H2O2 production observed with intact sperm at high cell concentrations is attributed to reaction of H2O2 with the cells, possibly with the plasma membrane, which is lost after hypo-osmotic treatment. Rabbit spermatozoa have glutathione peroxidase and glutathione reductase activities, but these seem to play little role in removal of H2O2 generated. The rate at low cell concentration is taken to be the unperturbed rate. The sources of H2O2 production in rabbit spermatozoa have been tentatively resolved into a low-molecular-weight component, lost after amphotericin treatment, a mitochondrial component and a rotenone-insensitive component that has not been identified.

scholarly journals Carbonic Anhydrase II Associated with Plasma Membrane in a Human Pancreatic Duct Cell Line (CAPAN-1)

2001 ◽  
Vol 49 (8) ◽  
pp. 1045-1053 ◽  
Author(s):  
Laetitia Alvarez ◽  
Marjorie Fanjul ◽  
Nicholas Carter ◽  
Etienne Hollande
Keyword(s):  
Plasma Membrane ◽  
Carbonic Anhydrase ◽  
Pancreatic Duct ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Brefeldin A ◽  
Duct Cell ◽  
Carbonic Anhydrase Ii ◽  
Apical Plasma Membrane ◽  
Cytosolic Side

The subcellular distribution of carbonic anhydrase II, either throughout the cytosol or in the cytoplasm close to the apical plasma membrane or vesicular compartments, suggests that this enzyme may have different roles in the regulation of pH in intra- or extracellular compartments. To throw more light on the role of pancreatic carbonic anhydrase II, we examined its expression and subcellular distribution in Capan-1 cells. Immunocytochemical analysis by light, confocal, and electron microscopy, as well as immunoblotting of cell homogenates or purified plasma membranes, was performed. A carbonic anhydrase II of 29 kD associated by weak bonds to the inner leaflet of apical plasma membranes of polarized cells was detected. This enzyme was co-localized with markers of Golgi compartments. Moreover, the defect of its targeting to apical plasma membranes in cells treated with brefeldin A was indicative of its transport by the Golgi apparatus. We show here that a carbonic anhydrase II is associated with the inner leaflet of apical plasma membranes and with the cytosolic side of the endomembranes of human cancerous pancreatic duct cells (Capan-1). These observations point to a role for this enzyme in the regulation of intra- and extracellular pH. (J Histochem Cytochem 49:1045–1053, 2001)

Subcellular Distribution of Low Molecular Weight Guanosine Triphosphate-Binding Proteins in Adipocytes: Colocalization with the Glucose Transporter Glut 4*

Endocrinology ◽  
1991 ◽  
Vol 129 (6) ◽  
pp. 3343-3350 ◽  
Author(s):  
MIREILLE CORMONT ◽  
JEAN-FRANCOIS TANTI ◽  
THIERRY GREMEAUX ◽  
EMMANUEL VAN OBBERGHEN ◽  
YANNICK LE MARCHAND-BRUSTEL
Keyword(s):  
Molecular Weight ◽  
Subcellular Distribution ◽  
Binding Proteins ◽  
Glucose Transporter ◽  
Low Molecular Weight ◽  
Guanosine Triphosphate ◽  
Glut 4
Sign in / Sign up

Export Citation Format

Share Document