Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[α-32P]ATP

1986 ◽  
Vol 64 (1) ◽  
pp. 13-20 ◽  
Author(s):  
Denis LeBel ◽  
Marlyne Beattie
Keyword(s):  
Binding Sites ◽  
Divalent Cations ◽  
Photoaffinity Labeling ◽  
Competitive Inhibitor ◽  
Atp Binding ◽  
Zymogen Granule ◽  
Atp Diphosphohydrolase ◽  
High Affinity ◽  
Granule Membrane ◽  
Triton X

Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[α-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 μM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 μM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A–Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-β-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.

scholarly journals Identification of the angiotensin II receptor in rat mesenteric artery

1984 ◽  
Vol 223 (3) ◽  
pp. 659-671 ◽  
Author(s):  
J McQueen ◽  
G D Murray ◽  
P F Semple
Keyword(s):  
Angiotensin Ii ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Divalent Cations ◽  
Target Tissue ◽  
Angiotensin Ii Receptor ◽  
High Affinity ◽  
Rat Mesentery ◽  
Rat Mesenteric Artery

Specific binding sites of high affinity and low capacity for 125I-angiotensin II have been identified in a membrane fraction derived from arterial arcades of the rat mesentery. Heterogeneity of binding sites and extensive tracer degradation necessitated the use of nonlinear regression methods for the analysis of radioligand binding data. Forward and reverse rate constants for the high affinity sites obtained by three experimental approaches were in good agreement and gave a dissociation equilibrium constant (Kd) of 19-74 pM (95% confidence interval). Affinities for a number of angiotensin-related peptides calculated from competitive binding curves were in the order 125I-angiotensin II = angiotensin II greater than angiotensin III greater than [Sar1,Ile8]angiotensin II greater than [Sar1,Gly8]angiotensin II. Angiotensin I and biochemically unrelated peptides had virtually no effect on binding of tracer angiotensin II. The divalent cations Mn2+, Mg2+ and Ca2+ stimulated 125I-angiotensin II binding at concentrations of 2-10 mM, as did Na+ at 50-100 mM. In the presence of Na+ or Li+, K+ had a biphasic effect. The chelating agents EDTA and EGTA were inhibitory, as were the thiol reagents dithiothreitol and cysteine. This study defined angiotensin II binding sites in a vascular target tissue of sufficiently high affinity to interact rapidly with plasma angiotensin II at physiological concentrations.

SOLUBILIZATION AND PARTIAL CHARACTERIZATION OF THYROID MEMBRANE TSH BINDING PROTEINS

1979 ◽  
Vol 92 (3) ◽  
pp. 512-521 ◽  
Author(s):  
B. Czarnocka ◽  
J. Nauman ◽  
G. Adler ◽  
W. Kiełczyński
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Gel Filtration ◽  
Soluble Proteins ◽  
The Other ◽  
High Affinity ◽  
Triton X ◽  
Maximal Capacity

ABSTRACT Crude plasma membranes obtained from bovine thyroids were found to possess one class of high affinity, low capacity binding sites for TSH with average association constant (Ka) of 1.301 × 109 m−1 and maximal capacity 8.76 × 10−10 m/mg of protein. Treatment of crude membranes fraction with 0.1 % Triton X-100 and the subsequent sonication in ultrasonic disintegrator resulted in solubilization of membranes proteins with mean recovery of 40.0 ± 6.2 %. Soluble proteins retained the property to bind [125I]TSH, but the binding of the hormone was decreased. The removal of the detergent from the solubilizate by gel filtration on Sephadex LH-20 increased the binding of TSH well above that demonstrated for crude thyroid membranes. The chromatography of soluble proteins on Ultrogel AcA-44 revealed the presence of two TSH binding proteins, one with the molecular weight (m.w.) above 130 000 daltons and the other with the m.w. approximately 30 000 daltons. The electrofocusing of solubilizate on Ampholine resulted in two protein peaks, one at pH 4.0–4.1 and the other at pH 4.4–4.6. The latter peak was shown to bind [125I]TSH specifically. The present results have confirmed the heterogeneous character of solubilized TSH receptor preparation and have shown that the hormone binding sites belong to acid proteins.

Effects of monovalent and divalent cations and of guanine nucleotides on binding of vasopressin to the rat mesenteric vasculature

1987 ◽  
Vol 65 (6) ◽  
pp. 1171-1181 ◽  
Author(s):  
Richard Larivière ◽  
Ernesto L. Schiffrin
Keyword(s):  
Rate Constant ◽  
Binding Sites ◽  
Divalent Cations ◽  
Dissociation Rate ◽  
Guanine Nucleotides ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Receptor Affinity ◽  
High Affinity ◽  
Binding Curves

The rat mesenteric vasculature contains high affinity binding sites specific for [3H]Arg8-vasopressin which mediate its vasoconstrictor action. We have investigated the in vitro effect of monovalent and divalent cations and guanine nucleotides on the interactions between [3H]Arg8-vasopressin and its receptor in this preparation. Binding was increased by divalent cations from fourfold in the presence of Mg2+ at 5 mM to ninefold in the presence of Mg2+ at 5 mM. The potency order of divalent cations to increase binding was Mn2+ > Co2+ > Ni2+ > Mg2+ > Ca2+ ≈ control without cations. Addition of Na+ or other monovalent cations (K+, Li+, and NH4+) in the presence or absence of divalent cations reduced binding significantly. Analysis of saturation binding curves showed a single high affinity site. In the presence of 5 mM Mn2+, binding capacity (Bmax) increased to 139 ± 23 fmol/mg protein. Receptor affinity was enhanced (KD decreased to 0.33 ± 0.07 nM). In presence of 5 mM Mg2+ or 150 mM Na+, fmax and affinity were reduced. The addition of 100 μM GTP or its nonhydrolyzable analogue, Gpp(NH)p, reduced receptor affinity in the presence of Mn2+ + Na+, Mg2+, and Mg2+ + Na+, but not in the presence of Mn2+ alone. Computer modeling of competition binding curves demonstrated that in contrast with saturation studies, the data were best explained by a two-site model with high affinity, low capacity sites and low affinity, high capacity sites. Mn2+ or Mn2+ + Na+ with or without guanine nucleotides resulted in a predominance of high affinity sites. GTP or Gpp(NH)p in the presence of Mg2+ or Mg2+ + Na+ induced a reduction of affinity of the high affinity binding sites and the number of these sites. In the presence of Mg2+ + Na+ and guanine nucleotides, high affinity sites were maximally decreased. An association kinetic study indicated that the association rate constant (K+1) was increased by divalent cations and reduced by guanine nucleotides, without change in the dissociation rate constant (K−1). The equilibrium dissociation constant (KD) calculated with these rate constants (K−1/K+1) was similar to that obtained in saturation experiments at steady state. Dissociation kinetics were biphasic, indicating the presence of two receptor states, one of high and one of low affinity, associated with a slow and a rapid dissociation rate. Cations and guanine nucleotides interact with one or more sites closely associated with vasopressin receptors, including possibly with a GTP-sensitive regulatory protein, to modulate receptor affinity for vasopressin.

Enhancement of [3H]DAGO1 binding to rat brain by low concentrations of monovalent cations

1987 ◽  
Vol 65 (11) ◽  
pp. 2338-2345 ◽  
Author(s):  
Gordon T. Bolger ◽  
Kendall A. Marcus ◽  
Ronald Thibou ◽  
Phil Skolnick ◽  
Ben Avi Weissman
Keyword(s):  
Rat Brain ◽  
Binding Sites ◽  
Divalent Cations ◽  
Opiate Receptors ◽  
Opiate Receptor ◽  
Affinity Binding ◽  
High Affinity ◽  
Regulatory Effects ◽  
In Vitro Effects

The effects of mono- and di-valent cations and the nonhydrolyzable guanyl nucleotide derivative 5′-guanylimidodiphosphate (Gpp(NH)p) on the binding of the selective, high affinity μ-opiate receptor agonist, [3H]DAGO ([3H]Tyr-D-Ala-Gly-Mephe-Gly-ol), to rat brain membranes were studied in a low ionic strength 5 mM Tris–HCl buffer. Na+ and Li+ (50 mM) maximally increased [3H]DAGO binding (EC50 values for Na+,2.9 mM and Li, 6.2 mM) by revealing a population of low affinity binding sites. The density of high affinity [3H]DAGO binding sites was unaffected by Na+ and Li+, but was maximally increased by 50 mM K+ and Rb+ (EC50 values for K+, 8.5 mM and Rb+, 12.9 mM). Divalent cations (Ca2+, Mg2+; 50 mM) inhibited [3H]DAGO binding. Gpp(NH)p decreased the affinity of [3H]DAGO binding, an effect that was enhanced by Na+ but not by K+. The binding of the μ-agonist [3H]dihydromorphine was unaffected by 50 mM Na+ in 5 mM Tris–HCl. In 50 mM Tris–HCl, Na+ (50 mM) inhibited [3H]DAGO binding by decreasing the density of high affinity binding sites and promoting low affinity binding. The effects of Na+ in 5 mM and 50 mM Tris–HCl were also investigated on the binding of other opiate receptor agonists and antagonists. [3H]D-Ala-D-Leu-enkephalin binding was increased and inhibited, [3H]etorphine binding increased and was unchanged, and both [3H]bremazocine and [3H]naloxone binding increased by 50 mM Na+ in 5 mM and 50 mM Tris–HCl, respectively. These findings indicate that the in vitro effects of Na+ at μ- and possibly other opiate receptors in rat brain are dependent on the concentration of Tris–HCl used in the assay buffer, lower concentrations of Tris-HCl revealing novel regulatory effects for Na+ at μ-opiate receptors.

scholarly journals Purification of pancreas type-I ATP diphosphohydrolase and identification by affinity labelling with the 5′-p-fluorosulphonylbenzoyladenosine ATP analogue

1995 ◽  
Vol 312 (2) ◽  
pp. 351-356 ◽  
Author(s):  
J Sévigny ◽  
Y P Côté ◽  
A R Beaudoin
Keyword(s):  
Specific Activity ◽  
Biochemical Properties ◽  
Zymogen Granule ◽  
Type I ◽  
Strong Reaction ◽  
Significant Similarity ◽  
Atp Diphosphohydrolase ◽  
Granule Membrane ◽  
Affinity Labelling ◽  
Molecular Masses

The enzyme recently identified as type-I ATP diphosphohydrolase (ATPDase; EC 3.6.1.5) has been purified from the zymogen granule membrane of pig pancreas. After solubilization with Triton X-100 and chromatographies on ion-exchange and Affi-Gel Blue columns an approximate 3500-fold purification was obtained. The enzyme preparation with a specific activity of 45 units/mg of protein was much further purified by PAGE under non-denaturing conditions. The active band localized on the gel contained two proteins after SDS/PAGE and silver staining, corresponding to apparent molecular masses of 56 and 54 kDa. The identity of the ATPDase was confirmed by an affinity labelling technique with 5′-p-fluorosulphonylbenzoyladenosine (FSBA) as an ATP analogue. The latter was detected by a Western blot technique. A strong reaction was observed with the band corresponding to 54 kDa. N-terminal sequence analysis revealed that the 56 kDa protein has significant similarities (50-72%) with lipases, whereas the 54 kDa enzyme has no significant similarity with any known proteins. N-glycosidase F treatment confirmed the glycoprotein nature of the enzyme and suggested that the enzyme bears several N-glycosylation sites. Comparisons of molecular masses and biochemical properties show that this ATPDase is different from other reported mammalian ATPDases.

Interaction of Factor V and Factor Va with Platelets

1979 ◽  
Author(s):  
P.B. Tracy ◽  
J. M. Peterson ◽  
M.E. Nesheim ◽  
F.C. McDuffie ◽  
K. G. Mann
Keyword(s):  
Binding Sites ◽  
Intrinsic Factor ◽  
Factor V ◽  
Single Chain ◽  
Apparent Dissociation Constant ◽  
Platelet Factor ◽  
High Affinity ◽  
Factor Va ◽  
Thrombin Activation ◽  
Triton X

We have used homogeneous single chain bovine factor V to examine the binding of both factor V and factor Va to bovine platelets, as well as to develop a double-antibody radioimmunoassay (RIA) to measure intrinsic platelet factor V. Reaction of the protein with 125I Bolton-Hunter reagent produced a labelled product which retained 90% of its cofactor activity and gave products indistinguishable from native factor V following thrombin activation. When incubated separately with washed bovine platelets, both 125I-factor V and Va underwent saturable and exchangeable binding. There are high affinity binding sites to which 500-900 V(Va) molecules are bound per platelet with an apparent dissociation constant of 3 χ 10-10 M, as well as binding sites of slightly lower affinity (Kd = 3 χ 10-9M) to which as many as 3500 V (Va) molecules are bound per platelet. Thrombin pretreatment of the platelets was not required for the binding of either factor V or Va. The RIA data for Triton X-100 lysed, washed bovine platelets revealed that 400-1000 intrinsic factor V molecules were present per platelet. Factor V clotting assays produced results consistent with the RIA data. These studies suggest that the factor V molecules intrinsic to the platelet are equivalent to the number of high affinity factor V (Va) binding sites present on the platelet memhrane surface. (Supported by Grant HL-17430 and the hayo Foundation).

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