scholarly journals The ionogenic nature of the secretory-granule membrane. Electrokinetic properties of isolated chromaffin granules

1972 ◽  
Vol 130 (3) ◽  
pp. 825-832 ◽  
Author(s):  
E. K. Matthews ◽  
R. J. Evans ◽  
P. M. Dean
Keyword(s):  
Surface Charge ◽  
Negative Charge ◽  
Single Type ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Granule Membrane ◽  
Dose Dependent Fashion ◽  
Ph Dependent ◽  
Dose Dependent ◽  
Anionic Surface

1. Chromaffin granules isolated from the bovine adrenal medulla possess an electrophoretic mobility of -1.12μm·s-1·cm·V-1, corresponding to a surface ζ potential of -14.4mV and surface charge density of 1.38×10-6C·cm-2. 2. The mobility of chromaffin granules is pH-dependent, indicating an amphoteric surface with an isoelectric point at pH3.0 and acidic groups with a pKa of 3.11. 3. Addition of bi- and ter-valent cations decreased the mobility of chromaffin granules in a dose-dependent fashion with a relative potency of La3+»Mn2+>Ca2+ >Sr2+>Mg2+>Ba2+. 4. Treatment with neuraminidase decreased the mobility of erythrocytes by 84%, whereas chromaffin-granule mobility was decreased by only 14%. This correlates well with the small complement of neuraminic acid present in the granule membrane. 5. The nature, origin and significance of the anionic surface charge of the chromaffin granule is discussed. It is concluded that the net negative charge at the surface of shear derives chiefly from a single type of chemical group, namely -CO2-, contributed by the α-carboxyl group of constituent proteins, the phospholipid phosphatidylserine and, to a lesser extent, the sialic acid component of glycoproteins.

scholarly journals Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes.

1992 ◽  
Vol 116 (2) ◽  
pp. 359-365 ◽  
Author(s):  
D Scheuner ◽  
C D Logsdon ◽  
R W Holz
Keyword(s):  
Chromaffin Cells ◽  
Secretory Vesicle ◽  
Xenopus Laevis Oocytes ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Vesicle Membrane ◽  
Sequence Of Events ◽  
Dopamine Beta Hydroxylase ◽  
Granule Membrane ◽  
Adrenal Chromaffin

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.

scholarly journals Phosphatidylinositol kinase. A component of the chromaffin-granule membrane

1973 ◽  
Vol 136 (3) ◽  
pp. 579-587 ◽  
Author(s):  
John H. Phillips
Keyword(s):  
Activation Energy ◽  
Initial Reaction ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Ph Profile ◽  
Phosphatidylinositol Kinase ◽  
Cytoplasmic Surface ◽  
Granule Membrane ◽  
Kinase A

Phosphorylation of bovine chromaffin granules by ATP leads to the formation of diphosphoinositide in the granule membrane. Both phosphatidylinositol kinase and its substrate are components of this membrane, and triphosphoinositide is not formed under the conditions of the assay. The reaction is Mg2+-dependent and is stimulated by Mn2+and F−ions. The initial reaction is rapid, with a broad pH profile and a ‘transition’ temperature for its activation energy at 27°C. The apparent Km for ATP is 5μm. ATP, N-ethylmaleimide, Cu2+ions and NaIO4 are inhibitory. The phospholipids of chromaffin-granule membranes have been analysed: 6.8% of the lipid P is found in phosphatidylinositol, and only 2–3% in phosphatidylserine. Comparison of the rate of phosphorylation of intact and lysed granules suggests that the sites for phosphorylation are on the outer (cytoplasmic) surface of the granules, and diphosphoinositide may therefore make an important contribution to the charge of the chromaffin granule in vivo.

Charges on the surfaces of two chlorites

Clay Minerals ◽  
1981 ◽  
Vol 16 (4) ◽  
pp. 347-359 ◽  
Author(s):  
Angela A. Jones
Keyword(s):  
Surface Charge ◽  
Surface Area ◽  
Negative Charge ◽  
Total Surface Area ◽  
Acid System ◽  
Negative Surface ◽  
Negative Surface Charge ◽  
Ph Dependent ◽  
Positively Charged

AbstractTwo chlorites, sheridanite and clinochlore, have been examined to determine their surface charge characteristics. In order to increase their surface area and to produce a measurable surface charge, the chlorites were treated with 10−3m, 10−2m, and 10−1m HCl in 10−2m MgCl2 solutions. These treatments are shown not to alter the crystallinity of the chlorites and to produce a small pH-dependent negative charge which is not directly related to the total surface area. The clinochlore, (Si6.13Al1.84)(Al1.53FeIII0.53FeII0.18Mg9.52)O20(OH)16, is more readily attacked by the acids than the sheridanite, (Si5.43Al2.55)(Al2.90FeII0.05Mg8.86)O20(OH)16, and also produces material with greater surface area and pH-dependent, negative, surface charge. It is concluded that: (i) isomorphous substitutions in the lattice are not reflected in a permanent surface charge; (ii) the observed surface charge arises not only at the edges of the particles but also at points where the chlorite is predisposed to attack by acids; (iii) in the chlorite-acid system used, anions—probably mainly silicate—block positively charged sites.

The membrane of the chromaffin granule

1971 ◽  
Vol 261 (839) ◽  
pp. 293-303 ◽  
Keyword(s):  
Membrane Fusion ◽  
Adrenal Medulla ◽  
Single Unit ◽  
Morphological Characteristics ◽  
Chromaffin Granules ◽  
Unit Membrane ◽  
Chromaffin Granule ◽  
Granule Membrane ◽  
Microsomal Membranes ◽  
Biochemical Analyses

Chromaffin granules of the adrenal medulla are surrounded by a single unit membrane. So far no special morphological characteristics of these membranes have been described. However, biochemical analyses have revealed the special properties of these membranes. The lipids are characterized by a high content of lysolecithin. It has been suggested that this specifically localized phospholipid is essential for the secretion of catecholamines, which involves membrane fusion. The proteins of the granule membrane have also been investigated. Two major components appear to be specific for chromaffin granules of several species. Three enzymes, namely an Mg 2+ -activated ATPase, dopamine β-hydroxylase and cytochrome b-559, are also known to be present in the granule membranes. The membranes of these organelles have no common structural backbone with microsomal membranes.

scholarly journals Ion permeability of isolated chromaffin granules.

1976 ◽  
Vol 68 (6) ◽  
pp. 601-631 ◽  
Author(s):  
R G Johnson ◽  
A Scarpa
Keyword(s):  
Time Course ◽  
Choline Chloride ◽  
Catecholamine Release ◽  
Ionophore A23187 ◽  
Chromaffin Granules ◽  
Ion Permeability ◽  
Chromaffin Granule ◽  
Internal Ph ◽  
Apparent Concentration ◽  
Granule Membrane

The passive ion permeability, regulation of volume, and internal pH of isolated bovine chromaffin granules were studied by radiochemical, potentiometric, gravimetric, and spectrophotometric techniques. Chromaffin granules behave as perfect osmometers between 340 and 1,000 mosM in choline chloride, NaCl, and KCl as measured by changes in absorbance at 430 nm or from intragranular water measurements using 3H2O and [14C]polydextran. By suspending chromaffin granules in iso-osmotic media of various metal ions and selectively increasing the permeability to either the cation or the anion by intrinsically permeable ions or specific ionophores, it was possible to determine by turbidity and potentiometric measurements the permeability to the counterion. These measurements indicate that the chromaffin granule is impermeable to the cations tested (Na+, K+, and H+). Limited H+ permeability across the chromaffin granule membrane was also shown by means of the time course of pH re-equilibration after pulsed pH changes in the surrounding media. The measurement of [14C]methylamine distribution indicates that a significant deltapH exists across the membrane, inside acidic, which at an external value of 6.85 has a value of 1.16. The deltapH is relatively insensitive to changes in the composition of the external media and can be enhanced or collapsed by the addition of ionophores and uncouplers. Measurement at various values of external pH indicates an internal pH of 5.5. Use of the ionophore A23187 indicates that Ca++ and Mg++ can be accumulated against an apparent concentration gradient with calcium uptake exceeding 50 nmol/mg of protein at saturation. These measurements also show that Ca++ and Mg++ are impermeable. Measurement of catecholamine release under conditions where intravesicular calcium accumulation is maximal indicates that catecholamine release does not occur. The physiological significance of the high impermeability to ions and the existence of a large deltapH are discussed in terms of regulation of uptake, storage, and release of catecholamines in chromaffin granules.

scholarly journals Passive ion permeability of the chromaffin-granule membrane

1977 ◽  
Vol 168 (2) ◽  
pp. 289-297 ◽  
Author(s):  
J H Phillips
Keyword(s):  
Osmotic Pressure ◽  
Complex Mixture ◽  
Low Permeability ◽  
Chromaffin Granules ◽  
Ion Permeability ◽  
Chromaffin Granule ◽  
Passive Permeability ◽  
Granule Membrane ◽  
Granule Matrix ◽  
Permeant Anion

‘Ghosts’ of bovine chromaffin granules, in which the complex mixture of proteins and solutes normally found in the granule matrix is replaced by buffered sucrose are osmotically sensitive. They shrink when the osmotic pressure of the suspension medium is increased, and swell if solute entry is facilitated by the addition of ionophores. Swelling in the presence of ionophores has been used to investigate the passive ion permeability of these membranes. They have a very low permeability to K+ ions (of the order of 10(-10) cm/s); their permeability to protons, Na+ and choline ions is too low to be detected by these methods. Their passive permeability to anions decreases in the order: CNS- greater than I- greater than CCl3CO2- greater than Br- greater than Cl- greater than SO4(2)- greater than CH3CO2-, HCO3-, F-, PO4(3)- the permeability to hiocyanate being of the order of 10(-7) cm/s. Coupled proton and anion entry is extremely slow, except for weak acids. Fluoride, unexpectedly, also appears to enter rapidly when proton/K+ exchange is facilitated by nigericin. In the presence of K+ salts, nigericin, like valinomycin, induces lysis of intact granules, an effect that is not dependent on the presence of a permeant anion, but is dependent on the pH gradient across the membrane.

scholarly journals Analysis of the Effects of Cigarette Smoke on Staphylococcal Virulence Phenotypes

2015 ◽  
Vol 83 (6) ◽  
pp. 2443-2452 ◽  
Author(s):  
Elisa K. McEachern ◽  
John H. Hwang ◽  
Katherine M. Sladewski ◽  
Shari Nicatia ◽  
Carola Dewitz ◽  
...  
Keyword(s):  
Surface Charge ◽  
Cigarette Smoke ◽  
Bacterial Infections ◽  
Content Type ◽  
Immune Resistance ◽  
Dose Dependent Fashion ◽  
Human Immune ◽  
Macrophage Killing ◽  
Dose Dependent

Cigarette smoking is the leading preventable cause of death, disease, and disability worldwide. It is well established that cigarette smoke provokes inflammatory activation and impairs antimicrobial functions of human immune cells. Here we explore whether cigarette smoke likewise affects the virulence properties of an important human pathogen,Staphylococcus aureus, and in particular methicillin-resistantS. aureus(MRSA), one of the leading causes of invasive bacterial infections. MRSA colonizes the nasopharynx and is thus exposed to inhalants, including cigarette smoke. MRSA exposed to cigarette smoke extract (CSE-MRSA) was more resistant to macrophage killing (4-fold higher survival;P< 0.0001). CSE-MRSA demonstrated reduced susceptibility to cell lysis (1.78-fold;P= 0.032) and antimicrobial peptide (AMP) (LL-37) killing (MIC, 8 μM versus 4 μM). CSE modified the surface charge of MRSA in a dose-dependent fashion, impairing the binding of particles with charge similar to that of AMPs by 90% (P< 0.0001). These changes persisted for 24 h postexposure, suggesting heritable modifications. CSE exposure increased hydrophobicity by 55% (P< 0.0001), which complemented findings of increased MRSA adherence and invasion of epithelial cells. CSE induced upregulation ofmprF, consistent with increased MRSA AMP resistance.S. aureuswithoutmprFhad no change in surface charge upon exposure to CSE.In vivo, CSE-MRSA pneumonia induced higher mouse mortality (40% versus 10%) and increased bacterial burden at 8 and 20 h postinfection compared to control MRSA-infected mice (P< 0.01). We conclude that cigarette smoke-induced immune resistance phenotypes in MRSA may be an additional factor contributing to susceptibility to infectious disease in cigarette smokers.

Plasminogen Interactions with Immobilized Fibrinogen

1989 ◽  
Vol 62 (04) ◽  
pp. 1078-1082 ◽  
Author(s):  
Burt Adelman ◽  
Patricia Ouynn
Keyword(s):  
Immunosorbent Assay ◽  
Aminocaproic Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Regions ◽  
Dose Dependent Fashion ◽  
Epsilon Aminocaproic Acid ◽  
Dose Dependent

SummaryThis report describes the binding of plasminogen to fibrinogen adsorbed onto polystyrene wells. Binding was determined by enzyme linked immunosorbent assay. Both glu- and lys-plasminogen bound to immobilized fibrinogen in a dose-dependent fashion. However, more lys- than glu-plasminogen bound when equal concentrations of either were added to immobilized fibrinogen. Plasminogen binding was inhibited by epsilon aminocaproic acid indicating that binding was mediated via lysine-binding regions of plasminogen. Soluble fibrinogen added in excess of immobilized fibrinogen did not compete for plasminogen binding but fibrinogen fragments produced by plasmin digestion of fibrinogen did. Treatment of immobilized fibrinogen with thrombin caused a small but significant (p <0.01) increase in plasminogen binding. These studies demonstrate that immobilized fibrinogen binds both glu- and lys-plasminogen and that binding is mediated via lysine-binding regions. These interactions may facilitate plasminogen binding to fibrinogen adsorbed on to surfaces and to cells such as platelets which bind fibrinogen.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

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