scholarly journals Inorganic pyrophosphate is located primarily in the mitochondria of the hepatocyte and increases in parallel with the decrease in light-scattering induced by gluconeogenic hormones, butyrate and ionophore A23187

1988 ◽  
Vol 254 (2) ◽  
pp. 379-384 ◽  
Author(s):  
A M Davidson ◽  
A P Halestrap
Keyword(s):  
Light Scattering ◽  
Adenine Nucleotides ◽  
Hormone Treatment ◽  
Subcellular Fractionation ◽  
Liver Mitochondria ◽  
Cell Protein ◽  
Ionophore A23187 ◽  
Inorganic Pyrophosphate ◽  
Rat Liver Cells ◽  
The Mean

1. The effects of a variety of hormones on the PPi content and light-scattering of isolated rat liver cells was studied. 2. The basal PPi content was about 130 pmol/mg of cell protein, and increased after hormone addition, in parallel with a decrease in light-scattering which we have observed previously [Quinlan, Thomas, Armston & Halestrap (1983) Biochem. J. 214, 395-404]. 3. The mean increases in PPi content with the agonists shown (as pmol/mg of protein) were: 0.1 microM-glucagon, 25; 20 microM-phenylephrine, 30; 25 nM-vasopressin, 127; glucagon + phenylephrine, 115; glucagon + vasopressin, 382; 100 microM-ADP, 50; 15 microM-A23187, 72; 1 mM-butyrate, 80. 4. In the absence of extracellular Ca2+, vasopressin had little effect on either the PPi content or the light-scattering of hepatocytes. 5. The magnitude of the increase in PPi content correlated with that of the decrease in light-scattering irrespective of the stimulating agent, provided that the PPi did not exceed 300 pmol/mg of protein. Above this value little additional change in light-scattering was observed. 6. Subcellular fractionation showed that over 90% of the cellular PPi was intramitochondrial in both control and stimulated cells. 7. The data support the conclusions of previous experiments using isolated liver mitochondria [Davidson & Halestrap (1987) Biochem. J. 246, 715-723] that hormones increase the mitochondrial matrix volume through a Ca2+-induced rise in matrix [PPi]. 8. It is further proposed that this increase in mitochondrial [PPi] allows entry of ADP into the mitochondria in exchange for PPi and is therefore responsible for the increase in total mitochondrial adenine nucleotides observed after hormone treatment.

Platelet dense bodies: a quantitative microprobe analysis

1976 ◽  
Vol 20 (2) ◽  
pp. 441-457
Author(s):  
R.J. Skaer ◽  
J.P. Emmines ◽  
P.D. Peters
Keyword(s):  
Adenine Nucleotides ◽  
Lipid Extraction ◽  
Dry Weight ◽  
Atomic Ratio ◽  
Human Platelets ◽  
Control Mechanisms ◽  
Inorganic Pyrophosphate ◽  
Dense Bodies ◽  
The Mean ◽  
Chloroform Methanol

The electron microprobe shows that the dense bodies of human platelets have a mean P:Ca peak ratio of 1–2. After treatment with dry chloroform/methanol this falls to 0-89. These ratios vary slightly from patient to patient. The use of calcium and phosphorus standards enables these peak ratios to be converted to atomic ratios. The size of the phosphorus peak remaining after lipid extraction was given absolute terms with reference to the known quantities of adenine nucleotides and inorganic pyrophosphate in dense bodies. From the mean P:Ca atomic ratio of 1–76 the quantity of calcium in dense bodies was 0-6 mg/10(11) platelets or 2–97 mg Ca/g dry weight of platelets. This is within the published range for total platelet calcium. If all the phosphorus extracted by lipid solvents were phospholipid there would be 5–65 mg/10(11) platelets, and it would occupy most of the space inside dense bodies. The dense bodies of pig platelets contain both magnesium and calcium in a varying ratio to each other. These results are discussed in relation to control mechanisms that may influence aggregation.

scholarly journals Transport and oxidation of choline by liver mitochondria

1977 ◽  
Vol 166 (3) ◽  
pp. 571-581 ◽  
Author(s):  
D. D. Tyler
Keyword(s):  
Mitochondrial Membrane ◽  
Adenine Nucleotides ◽  
Close Correlation ◽  
Liver Mitochondria ◽  
Mitochondrial Swelling ◽  
Ionophore A23187 ◽  
Phospholipase Activity ◽  
Choline Transport ◽  
Tightly Coupled

1. Rapid choline oxidation and the onset of Pi-induced swelling by liver mitochondria, incubated in a sucrose medium at or above pH7.0, required the addition of both Pi and an uncoupling agent. Below pH7.0, Pi alone was required for rapid choline oxidation and swelling. 2. Choline oxidation was inhibited by each of several reagents that also inhibited Pi-induced swelling under similar conditions of incubation, including EGTA, mersalyl, Mg2+, the Ca2+-ionophore A23187, rotenone and nupercaine. None of these reagents had any significant effect on the rate of choline oxidation by sonicated mitochondria. There was therefore a close correlation between the conditions required for rapid choline oxidation and for Pi-induced swelling to occur, suggesting that in the absence of mitochondrial swelling the rate of choline oxidation is regulated by the rate of choline transport across the mitochondrial membrane. 3. Respiratory-chain inhibitors, uncoupling agents (at pH6.5) and ionophore A23187 caused a loss of endogenous Ca2+ from mitochondria, whereas nupercaine and Mg2+ had no significant effect on the Ca2+ content. Inhibition of choline oxidation and mitochondrial swelling by ionophore A23187 was reversed by adding Ca2+, but not by Mg2+. It is concluded that added Pi promotes the Ca2+-dependent activation of mitochondrial membrane phospholipase activity in respiring mitochondria, causing an increase in the permeability of the mitochondrial inner membrane to choline and therefore enabling rapid choline oxidation to occur. Nupercaine and Mg2+ appear to block choline oxidation and swelling by inhibiting phospholipase activity. 4. Choline was oxidized slowly by tightly coupled mitochondria largely depleted of their endogenous adenine nucleotides, suggesting that these compounds are not directly concerned in the regulation of choline oxidation. 5. The results are discussed in relation to the possible mechanism of choline transport across the mitochondrial membrane in vivo and the influence of this process on the pathways of choline metabolism in the liver.

scholarly journals The distribution and chemical nature of radioactive folates in rat liver cells and rat liver mitochondria

1979 ◽  
Vol 178 (3) ◽  
pp. 651-659 ◽  
Author(s):  
R J Cook ◽  
J A Blair
Keyword(s):  
Oral Administration ◽  
Rat Liver ◽  
Subcellular Fractionation ◽  
Liver Mitochondria ◽  
Liver Cells ◽  
Rat Liver Mitochondria ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Rat Liver Cells ◽  
Cell Cytosol

Subcellular fractionation of rat liver cells revealed that a mixture of 14C- and 3H-labelled folic acid was distributed approximately equally between the mitochondria and cytosol 2, 24, 48 and 72 h after oral administration. Subfractionation of liver mitochondria 48 h after oral administration showed that the radioactivity was mainly associated with the inner membrane (27.7%) and matrix (51.5%). Hot-ascorbate extraction of the cell cytosol, mitochondrial inner membrane and matrix showed the majority of folates were present as polyglutamates. Acid treatment of isolated folates from cytosol, inner membrane and matrix produced breakdown products consistent with scission of tetrahydrofolates. The folates isolated in the mitochondrial matrix were bound to protein that had an estimated mol. wt. of 90,000.

Swelling of Fresh and Aged Liver Mitochondria as affected by Succinate, Adenine Nucleotides and Thyroxine

Nature ◽  
1960 ◽  
Vol 186 (4724) ◽  
pp. 556-558 ◽  
Author(s):  
P. EMMELOT ◽  
C. J. BOS ◽  
P. J. BROMBACHER ◽  
I. H. M. REYERS
Keyword(s):  
Adenine Nucleotides ◽  
Liver Mitochondria

scholarly journals Control of Choline Oxidation in Liver Mitochondria by Adenine Nucleotides

1965 ◽  
Vol 240 (4) ◽  
pp. 1836-1842
Author(s):  
Tsuneo Kagawa ◽  
David R. Wilken ◽  
Henry A. Lardy
Keyword(s):  
Adenine Nucleotides ◽  
Liver Mitochondria

Analytical Subcellular Fractionation Studies on Rat Liver and on Isolated Jejunal Enterocytes with Special Reference to the Separation of Lysosomes, Peroxisomes and Mitochondria

1976 ◽  
Vol 50 (5) ◽  
pp. 355-366 ◽  
Author(s):  
T. J. Peters ◽  
H. Shio
Keyword(s):  
Rat Liver ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Low Molecular Weight ◽  
Rat Jejunum ◽  
Marker Enzymes ◽  
Good Separation ◽  
Subcellular Organelles ◽  
Rat Liver Cells ◽  
Isopycnic Centrifugation

1. Enterocytes were isolated from rat jejunum and characterized morphologically. 2. Attempts to separate the enterocyte subcellular organelles, characterized by their marker enzymes, with isopycnic centrifugation were unsuccessful but good separation of peroxisomes, lysosomes and mitochondria was achieved by sedimentation through a shallow sucrose density gradient with a superimposed inverse gradient of low-molecular-weight dextran. 3. The properties and enzyme activities of the principal subcellular organelles in rat liver cells and enterocytes were compared.

Light scattering by phonons in the presence of a heat flow

1968 ◽  
Vol 46 (24) ◽  
pp. 2843-2845 ◽  
Author(s):  
Allan Griffin
Keyword(s):  
Light Scattering ◽  
Heat Flow ◽  
Temperature Gradient ◽  
Free Path ◽  
Mean Free Path ◽  
Photon Scattering ◽  
The Mean ◽  
Anti Stokes

If the temperature in an insulating crystal decreases in the z-direction, there are more phonons with momentum qz > 0 than with qz < 0. The resulting difference between the Stokes and anti-Stokes Brillouin intensities is proportional to the mean free path of the phonon involved and to the temperature gradient. The effect should be observable by either neutron or photon scattering.

scholarly journals Identification of high-affinity (Kd 0.35 mumol/L) and low-affinity (Kd 7.9 mumol/L) platelet binding sites for ADP and competition by ADP analogues

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 110-116 ◽  
Author(s):  
JR Jefferson ◽  
JT Harmon ◽  
GA Jamieson
Keyword(s):  
Platelet Activation ◽  
Dissociation Constants ◽  
Partial Agonist ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Ionophore A23187 ◽  
High Affinity ◽  
Binding Isotherms ◽  
High Affinity Site ◽  
High Concentrations

Steady-state binding of ADP to blood platelets and isolated membranes has not previously been obtained because of complications arising from metabolism of the ligand and dilution due to its secretion from storage granules. In the present studies, competition binding isotherms (n = 9) using paraformaldehyde-fixed platelets showed that [2–3 H]ADP bound to two sites with a small amount (approximately 5% of total) of nonspecific binding: 410,000 +/- 40,000 sites of low affinity (Kd 7.9 +/- 2.0 mumol/L) and 160,000 +/- 20,000 sites of high affinity (Kd 0.35 +/- 0.04 mumol/L) corresponding to the ADP concentration required for activation in fresh platelets (0.1–0.5 mumol/L). All agonists and antagonists examined were able to compete with ADP at the high-affinity site. The strong platelet agonists 2-methylthio ADP and 2-(3- aminopropylthio)ADP competed with ADP at the high-affinity site with dissociation constant values of 7 mumol/L and 200 mumol/L, respectively. The partial agonist 2′,3′-dialdehyde ADP and the weak agonist GDP also competed at the high-affinity site with Kd values of 5 mumol/L and 49 mumol/L, respectively. The sequence of binding affinities of other adenine nucleotides at the high-affinity site corresponded to their relative activities as known antagonists of platelet activation by ADP; namely, ADP(Kd 0.35 mumol/L) approximately equal to ATP (Kd 0.45 mumol/L) much greater than AMP (Kd 360 mumol/L). Adenosine and 2-chloroadenosine did not compete with ADP. ADP binding to the high-affinity site was inhibited by p-mercuribenzene sulfonate (Ki 250 mumol/L) but only very weakly by 5′-p- fluorosulfonylbenzoyladenosine (Ki 1 mmol/L). All the above nucleotides also competed with ADP at the low-affinity sites but, because of the high concentrations of competing nucleotide required, dissociation constants at this site were obtained only for ATP (21 mumol/L), 2-MeS ADP (200 mumol/L) and 2′,3′-dialdehyde ADP (270 mumol/L). 8-Bromo ADP competed strongly with ADP at the high-affinity site (Kd 0.40 mumol/L) but weakly if at all at the low-affinity site. 8-Bromo ADP inhibited platelet activation induced by ADP (EC50 approximately 100 mumol/L) but not by collagen, thrombin, or ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS).

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