Fine-Structural Localization of Adenosine Triphosphatase in the Rectum of Calliphora

1968 ◽  
Vol 3 (1) ◽  
pp. 17-32
Author(s):  
M. J. BERRIDGE ◽  
B. L. GUPTA
Keyword(s):  
Plasma Membrane ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Fluid Transport ◽  
Microsomal Fraction ◽  
Adenosine Diphosphate ◽  
Structural Basis ◽  
Osmotic Gradient ◽  
Ph Optimum ◽  
Structural Localization

Adenosine triphosphatase (ATPase) activity in the rectal papillae of Calliphora has been studied by biochemical and histochemical techniques. The microsomal fraction contained a Mg2+-activated ATPase with a pH optimum of 8.0. The enzyme was not stimulated by the addition of Na+ plus K+ and was insensitive to ouabain. Histochemical studies using modifications of the Wachstein-Meisel method showed that at pH 7.2 this Mg2+-activated ATPase was specifically localized on the intracellular surface of the lateral plasma membranes. A similar though less intense reaction was obtained with adenosine diphosphate and inosine triphosphate, but not with guanosine triphosphate, uridine triphosphate or β-glycerophosphate as substrates. At an acid pH (6.6-6.8), very little reaction occurred on the lateral plasma membrane but some reaction product was present in mitochondria and nuclei. Very little enzyme activity was found in the flattened rectal epithelium. These results are discussed in relation to the available data on transport ATPases and on the structural basis of fluid transport by rectal papillae. It is proposed that the ATPase localized on the stacks of lateral plasma membrane may be involved with ion secretion into the intercellular spaces to create the osmotic gradient necessary to extract water from the lumen.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF Mg++-DEPENDENT DINITROPHENOL-STIMULATED ADENOSINE TRIPHOSPHATASE IN HUMAN BLOOD PLATELETS

1967 ◽  
Vol 15 (5) ◽  
pp. 267-272 ◽  
Author(s):  
VICTOR G. VETHAMANY ◽  
SYDNEY S. LAZARUS
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Human Blood ◽  
Adenosine Triphosphatase ◽  
Blood Platelets ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Structural Localization ◽  
Adenosine Triphosphatase Activity ◽  
Human Blood Platelets

Fine structural localization of adenosine triphosphatase activity was studied in human platelets briefly fixed in cold formol calcium and then incubated in lead medium with added dinitrophenol. Under these conditions, the Mg++-dependent dinitrophenol-stimulated adenosine triphosphatase of platelet mitochondria was demonstrated, but neither granules nor plasma membrane showed enzyme activity.

Biogenesis of Plasma Membranes in Salt Glands of Salt-Stressed Domestic Ducklings: Localization of Acyltransferase Activity

1972 ◽  
Vol 11 (3) ◽  
pp. 855-873
Author(s):  
A. M. LEVINE ◽  
JOAN A. HIGGINS ◽  
R. J. BARRNETT
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Salt Gland ◽  
Secretory Cells ◽  
Salt Glands ◽  
Acyltransferase Activity ◽  
Structural Localization ◽  
Differentiated Cells ◽  
Golgi Membranes

In response to salt water stress there is a marked increase in the plasma membranes of the epithelial secretory cells of the salt glands of domestic ducklings. In the present study, the fine-structural localization of the acyltransferases involved in synthesis of phospholipids has been investigated in this tissue during this increased biogenesis of plasma membranes. The specific activity of the acyltransferases of the salt gland rose in response to salt stress, and this preceded the rapid increase in weight and cellular differentiation. After the weight increase of the gland became established, the specific activity of the acyltransferases declined, but the total activity remained constant. Salt gland tissue fixed in a mixture of glutaraldehyde and formaldehyde retained 35% of the acyltransferase activity of unfixed tissue. Cytochemical studies of the localization of acyltransferase activity in fixed and unfixed salt gland showed reaction product associated only with the lamellar membranes of the Golgi complex. This localization occurred in partially differentiated cells from salt-stressed glands to the greatest extent; and to only a small extent in cells of control tissue from unstressed salt glands. Omission of substrates resulted in absence of reaction product in association with the Golgi membranes. In addition, vesicles having limiting membranes morphologically similar to the plasma membrane occurred between the Golgi region and the plasma membrane in the partially differentiated cells. The phospholipid component of the plasma membrane appears therefore to be synthesized in association with the Golgi membranes and the membrane packaged at this site from which it moves in the form of vesicles to fuse with the pre-existing plasma membrane.

scholarly journals Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum α-l-fucosidase from rat testis and epididymal spermatozoa

1996 ◽  
Vol 318 (3) ◽  
pp. 821-831 ◽  
Author(s):  
Manuel AVILÉS ◽  
Irene ABASCAL ◽  
José Angel MARTÍNEZ-MENÁRGUEZ ◽  
María Teresa CASTELLS ◽  
Sheri R. SKALABAN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Light And Electron Microscopy ◽  
Enzymic Activity ◽  
Epididymal Sperm ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Neutral Ph

1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum α-l-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for α-l-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the α-l-fucosidase activity was associated with the 48000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of α-l-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH–activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm–egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm–egg interactions.

scholarly journals Binding of Adenosine Diphosphate (ADP) to Isolated Human Platelet Membranes

1977 ◽  
Author(s):  
J. Lips ◽  
J. J. Sixma
Keyword(s):  
Binding Sites ◽  
Association Constant ◽  
Plasma Membranes ◽  
Human Platelet ◽  
Membrane Vesicles ◽  
Adenosine Diphosphate ◽  
Ph Optimum ◽  
High Affinity ◽  
Absolute Requirement ◽  
Millipore Filtration

Human platelet plasma membranes were isolated according to the glycerol loading technique of Barber and Jamieson. The binding of 14C ADP was studied with Millipore filtration in a Ca2+ and Mg2+ containing buffer at pH 7,4. At least two types of binding sites were found: A high affinity system with a maximum binding of 160 pMoles/mg protein and an association constant of 1,1 χ 106 M-1; and alow affinity system with a maximum binding of about 4500 pMoles/mg protein and an association constant of 0,6 χ 1θ4 M-1. The binding according to the high affinity system showed little temperature dependency (Q10 = 1,10). The pH optimum was at 7,3. Ca2+ ions were an absolute requirement for binding.Nucleoside diphosphokinase (NDPK) was found in the membrane vesicles. Evidence that this enzyme was not responsible for ADP binding was obtained. The enzyme is Mg2+ dependent and is inhibited by AMP, in contrast to ADP binding. The Q10 was 1,44.ADP binding was inhibited by ATP, IDP and β/γ-imidoadenosine triphosphate.

scholarly journals Flexibility of an Active Center in Sodium-Plus-Potassium Adenosine Triphosphatase

1969 ◽  
Vol 54 (1) ◽  
pp. 306-326 ◽  
Author(s):  
R. L. Post ◽  
S. Kume ◽  
T. Tobin ◽  
B. Orcutt ◽  
A. K. Sen
Keyword(s):  
Active Center ◽  
Adenosine Triphosphate ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Adenosine Diphosphate ◽  
Ion Concentration ◽  
Potassium Ions ◽  
Sodium Ions ◽  
Intact Cells ◽  
Electrophoretic Patterns

In plasma membranes of intact cells an enzymatic pump actively transports sodium ions inward and potassium ions outward. In preparations of broken membranes it appears as an adenosine triphosphatase dependent on magnesium, sodium, and potassium ions together. In this adenosine triphosphatase a phosphorylated intermediate is formed from adenosine triphosphate in the presence of sodium ions and is hydrolyzed with the addition of potassium ions. The normal intermediate was not split by adenosine diphosphate. However, selective poisoning by N-ethylmaleimide or partial inhibition by a low magnesium ion concentration yielded an intermediate split by adenosine diphosphate and insensitive to potassium ions. Pulse experiments on the native enzyme supported further a hypothesis of a sequence of phosphorylated forms, the first being made reversibly from adenosine triphosphate in the presence of sodium ion and the second being made irreversiblyfrom the first and hydrolyzed in the presence of potassium ion. The cardioactive steriod inhibitor, ouabain, appeared to combine preferentially with the second form. Phosphorylation was at the same active site according to electrophoretic patterns of proteolytic phosphorylated fragments of both reactive forms. It is concluded that there is a conformational change in the active center for phosphorylation during the normal reaction sequence. This change may be linked to one required theoretically for active translocation of ions across the cell membrane.

scholarly journals Heparan sulphate-degrading endoglycosidase in liver plasma membranes

1988 ◽  
Vol 250 (3) ◽  
pp. 719-726 ◽  
Author(s):  
J T Gallagher ◽  
A Walker ◽  
M Lyon ◽  
W H Evans
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Heparan Sulphate ◽  
Biologically Active ◽  
Cell Periphery ◽  
Cellular Interactions ◽  
Hydrophobic Membrane ◽  
Ph Optimum ◽  
Liver Plasma Membranes ◽  
Ph Range

An endoglycosidase is described in isolated liver plasma membranes that brings about a rapid and selective degradation of membrane-associated heparan sulphate, pre-labelled biosynthetically with Na2(35)SO4. The enzyme attacked mainly the polysaccharide chains of a hydrophobic membrane proteoglycan and it had little effect on a proteoglycan that could be displaced from the membranes with 1.0 M-NaCl. The highest activity was measured in the pH range 7.5-8.0, and the enzyme was almost completely inhibited below pH 5.5. Breakdown of susceptible polysaccharide chains was fast, being complete in 20-30 min. The major oligosaccharide fraction (Mr approx. 6000) produced by the enzyme was considerably smaller than the intact heparan sulphate chains. Enzyme activity was retained in membranes solubilized in 1% (v/v) Triton X-100. The high pH optimum and plasma-membrane association distinguish this enzyme from other heparan sulphate-degrading endoglycosidases that have acid pH optima and may be of lysosomal origin. A plasma-membrane endoglycosidase could modulate cellular interactions mediated by heparan sulphate, and/or release biologically active fragments of the polysaccharide from the cell periphery.

Specific binding of [3H]oxytocin in the female rat brain

1983 ◽  
Vol 61 (9) ◽  
pp. 989-995 ◽  
Author(s):  
B. M. Ferrier ◽  
S.A. McClorry ◽  
A. W. Cochrane
Keyword(s):  
Plasma Membrane ◽  
Limbic System ◽  
Microsomal Fraction ◽  
Specific Binding ◽  
Cell Types ◽  
Time Dependent ◽  
Female Rat ◽  
Ph Optimum ◽  
Rat Tissue ◽  
Different Cell Types

Because of demonstrated effects of oxytocin on some limbic system mediated behaviours, the specific binding of [3H]oxytocin to a plasma membrane containing fraction of rat limbic tissue has been studied. The binding of the microsomal fraction of estrogenized, female rat tissue was time dependent and saturable, with a Bmax of 2.5 × 10−l3 moles per milligram of protein and an apparent KD of 3.53 × 10−8 M, and appeared to show positive cooperativity. The pH optimum of the binding was 6.0, close to the pH optimum for oxytocin – neurophysin binding; however, other results show the two types of binding to be different. The microsomal fraction did not appreciably degrade oxytocin under the conditions used for [3H]oxytocin binding. The distribution in limbic tissue of oxytocin-degrading activity and of individual enzymes capable of degrading oxytocin has been examined and an interplay of enzymes concentrated in different cell types is proposed.

scholarly journals Requirement for guanosine triphosphate for cholera-toxin-catalysed incorporation of adenosine diphosphate ribose into rat liver plasma membranes and for activation of adenylate cyclase

1980 ◽  
Vol 186 (3) ◽  
pp. 749-754 ◽  
Author(s):  
C A Doberska ◽  
A J S MacPherson ◽  
B R Martin
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Adenosine Diphosphate ◽  
Activation Process ◽  
Liver Plasma Membrane ◽  
Adp Ribosylation ◽  
A Subunit

1. Cholera toxin was shown to require the presence of GTP to activate rat liver plasma-membrane adenylate cyclase. ATP did not affect the activation process. 2. Cholera toxin catalysed the incorporation of 32P from NAD labelled in the alpha-phosphate group of the ADP moiety into a rat liver plasma-membrane protein with a subunit mol.wt. of 42 500. This is taken to demonstrate ADP-ribosylation. The ADP-ribosylation of this protein also required GTP and was unaffected by ATP. 3. Nicotinamide inhibited both the activation of adenylate cyclase by cholera toxin and the ADP-ribosylation of the protein of 42 500 subunit mol wt. Neither the activation nor the ADP-ribosylation could be reversed by treatment with nicotinamide in the presence of cholera toxin.

scholarly journals Alkaline ribonuclease and phosphodiesterase activity in rat liver plasma membranes

1973 ◽  
Vol 132 (3) ◽  
pp. 449-458 ◽  
Author(s):  
Terence D. Prospero ◽  
Malcolm L. E. Burge ◽  
Kenneth A. Norris ◽  
Richard H. Hinton ◽  
Eric Reid
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Ribonuclease Activity ◽  
Nuclear Fraction ◽  
Phosphodiesterase Activity ◽  
Ph Optimum ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes

The ribonuclease and phosphodiesterase activities of rat liver plasma membranes, purified from the crude nuclear fraction by centrifugation in an A-XII zonal rotor and flotation, were examined and compared. The plasma membrane is responsible for between 65 and 90% of the phosphodiesterase activity of the cell and between 25 and 30% of the particulate ribonuclease activity measured at pH8.7 in the presence of 7.5mm-MgCl2. Both enzymes were most active between pH8.5 and 8.9. Close to the pH optimum, both enzymes were more active in Tris buffer than in Bicine or glycine buffer. Both plasma-membrane phosphodiesterase and ribonuclease were strongly activated by Mg2+, there being at least a 12-fold difference between the activity in the presence of Mg2+ and of EDTA. There is, however, a difference in the response of the enzymes to Mg2+ and EDTA in that the phosphodiesterase is fully activated by 1.0mm-MgCl2 and fully inhibited by 1.0mm-EDTA, whereas the ribonuclease requires 7.5mm-MgCl2 for full activation and 5mm-EDTA for full inhibition. Density-gradient centrifugation has indicated that on solubilization in Triton X-100 most of the ribonuclease activity is released into a small fragment of the same size as that containing the phosphodiesterase activity. The relationship between the two activities is discussed in view of these results.

scholarly journals Identification and subcellular localization of sphinganine-phosphatases in rat liver

1995 ◽  
Vol 311 (1) ◽  
pp. 139-146 ◽  
Author(s):  
P De Ceuster ◽  
G P Mannaerts ◽  
P P Van Veldhoven
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Water Soluble ◽  
Nuclear Fraction ◽  
Cell Fractionation ◽  
Acidic Ph ◽  
Ph Optimum ◽  
High Affinity ◽  
Neuronal Tissues

One of the primary products of [4,5-3H]sphinganine phosphate, added to fibroblast cultures, is sphinganine [Van Veldhoven and Mannaerts (1994) Biochem. J. 299, 597-601], implicating the physiological action of (a) hitherto unknown phosphatase(s). We have now further characterized this activity in rat liver. In homogenates, the dephosphorylation appeared to be catalysed by multiple enzymes. A low-affinity system was active at acidic pH, whereas at physiological pH values hydrolysis was carried out by a high-affinity enzyme. The latter was sensitive to Zn2+ and detergents and possessed a pH optimum of 7.5. Upon cell fractionation the major portion of the high-affinity activity was recovered in the nuclear and microsomal fractions. Further separation of the microsomal fraction showed an association predominantly with vesicles derived from the plasma membrane. Likewise, when plasma membranes were prepared from the nuclear fraction, the high-affinity phosphatase co-purified with the plasma membrane markers. From the differential effects of bivalent cations, chelators, water-soluble and amphiphilic phosphate esters, detergents and other compounds, it could be concluded that the plasma membrane-associated sphinganine-phosphatase activity is not due to alkaline phosphatase, dolichol-phosphatase, the N-ethylmaleimide-insensitive phosphatidate phosphatase or ceramide-phosphatase. The dephosphorylation observed at acidic pH in homogenates appeared also to be enriched in purified plasma membranes and might represent a side-activity of ceramide-phosphatase. We speculate that the high-affinity phosphatase, which is especially active in neuronal tissues, plays a role in the attenuation of bioactive phosphorylated sphingoid bases such as sphingenine phosphate, and propose to name it sphingosine-phosphatase.

Sign in / Sign up

Export Citation Format

Share Document