ALG-2 divalent-ion affinity: Calorimetric analysis of the des23 versions reveals high-affinity site for Mg2+

ABSTRACT Extracts of a multiply peptidase-deficient (pepNABDPQTE iadA iaaA) Salmonella enterica serovar Typhimurium strain contain an aspartyl dipeptidase activity that is dependent on Mn2+. Purification of this activity followed by N-terminal sequencing of the protein suggested that the Mn2+-dependent peptidase is DapE (N-succinyl-l,l-diaminopimelate desuccinylase). A dapE chromosomal disruption was constructed and transduced into a multiply peptidase-deficient (MPD) strain. Crude extracts of this strain showed no aspartyl peptidase activity, and the strain failed to utilize Asp-Leu as a leucine source. The dapE gene was cloned into expression vectors in order to overproduce either the native protein (DapE) or a hexahistidine fusion protein (DapE-His6). Extracts of a strain carrying the plasmid overexpresssing native DapE in the MPD dapE background showed a 3,200-fold elevation of Mn2+-dependent aspartyl peptidase activity relative to the MPD dapE+ strain. In addition, purified DapE-His6 exhibited Mn2+-dependent peptidase activity toward aspartyl dipeptides. Growth of the MPD strain carrying a single genomic copy of dapE on Asp-Leu as a Leu source was slow but detectable. Overproduction of DapE in the MPD dapE strain allowed growth on Asp-Leu at a much faster rate. DapE was found to be specific for N-terminal aspartyl dipeptides: no N-terminal Glu, Met, or Leu peptides were hydrolyzed, nor were any peptides containing more than two amino acids. DapE is known to bind two divalent cations: one with high affinity and the other with lower affinity. Our data indicate that the form of DapE active as a peptidase contains Zn2+ in the high-affinity site and Mn2+ in the low-affinity site.

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Cytochrome c Oxidase: Biphasic Kinetics Result from Incomplete Reduction of Cytochrome a by Cytochrome c Bound to the High-Affinity Site

Biochemistry ◽

10.1021/bi00031a023 ◽

1995 ◽

Vol 34 (31) ◽

pp. 10000-10008 ◽

Cited By ~ 8

Author(s):

Jaime Ortega-Lopez ◽

Neal C. Robinson

Keyword(s):

Cytochrome C ◽

Cytochrome C Oxidase ◽

Biphasic Kinetics ◽

High Affinity ◽

High Affinity Site

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Binding of the Peroxisomal Targeting Sequence SKL Is Specified by a Low-Affinity Site in Castor Bean Glyoxysomal Membranes (A Domain Next to the SKL Binds to a High-Affinity Site)

PLANT PHYSIOLOGY ◽

10.1104/pp.113.3.943 ◽

1997 ◽

Vol 113 (3) ◽

pp. 943-949 ◽

Cited By ~ 4

Author(s):

N. E. Wolins ◽

R. P. Donaldson

Keyword(s):

Castor Bean ◽

High Affinity ◽

High Affinity Site ◽

Peroxisomal Targeting

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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 ◽

10.1182/blood.v71.1.110.bloodjournal711110 ◽

1988 ◽

Vol 71 (1) ◽

pp. 110-116 ◽

Cited By ~ 1

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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A gonadotropin-releasing hormone type neuropeptide with a high affinity binding site for copper(ii) and nickel(ii)

Metallomics ◽

10.1039/c8mt00279g ◽

2019 ◽

Vol 11 (2) ◽

pp. 404-414 ◽

Cited By ~ 5

Author(s):

Kevin K. Tran ◽

Bhawantha M. Jayawardena ◽

Maurice R. Elphick ◽

Christopher E. Jones

Keyword(s):

Binding Site ◽

Gonadotropin Releasing Hormone ◽

Affinity Binding ◽

Asterias Rubens ◽

High Affinity Binding ◽

Releasing Hormone ◽

High Affinity ◽

Evolutionarily Conserved ◽

High Affinity Site ◽

High Affinity Binding Site

Gonadotropin releasing hormone from Asterias rubens binds Cu(ii) in a nitrogen-rich, high-affinity site. Cu(ii)-binding is an evolutionarily conserved feature of GnRH-type neuropeptides.

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ADP-BINDING SITES IN PLATELETS: CHARACTERIZATION BY PHOTOAFFINITY LABELING AND BINDING STUDIES WITH FIXED PLATELETS

10.1055/s-0038-1644463 ◽

1987 ◽

Author(s):

J R Jefferson ◽

J T Harmon ◽

G A Jamieson

Keyword(s):

Specific Binding ◽

Photoaffinity Labeling ◽

Binding Studies ◽

Dense Granules ◽

High Affinity ◽

High Affinity Site ◽

Adp Receptors ◽

Antagonists And Inhibitors ◽

Spacer Arm ◽

Formalin Fixed

Attempts to photoaffinity label platelet ADP receptors with 2-azidoADP have not been successful possibly due to the absence of a spacer arm between the nucleotide and the photolabile group. We have synthesized a probe having a long spacer arm by coupling 2-(3-aminopropylthio)-ADP to succinimidyl 4-3H-azidobenzoate. Labeling competable by ADP could not demonstrated with intact platelets. With isolated platelet membranes, three bands (Mr 140,000, 110,000 and 46,000) were labeled that were not competed by ADP while three other bands (Mr 188,000, 92,000 and 51,000) were competable by 100 uM ADP.Another problem in characterizing ADP receptors has been complications due to ADP metabolism and secretion from the dense granules. To avoid this problem we have measured the binding of ADP and analogues to formalin-fixed platelets. ADP bound to two sites (Kl, 0.35 ± 0.04 uM; R1, 160,000 ± 20,000 sites/platelet; K2 7.9 ± 2.0 uM; R2, 400,000 ± 40,000 sites/platelet) with low non-specific binding: these values are in agreement with ADP concentrations required for activation. Affinity at the high affinity site was in the sequence ADP(0.35 uM)=ATP(0.4 uM)›2-MeS.ADP(6.8 uM)› GDP(49 uM) › AMP(360 uM); adenosine did not compete. Binding at the high affinity site was blocked by pMBS (EC50 250 uM) and 5-fluoro-sulfonylbenzoyladenosine (EC50 1 mM). This is the first report of photoaffinity labeling of putative ADP receptors. Our experiments with fixed platelets suggest that they may be useful in testing agonists, antagonists and inhibitors in the absence of complications due to secretion and metabolism.

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The binding of [3H]adenosine to synaptosomal and other preparations from the mammalian brain

Biochemical Journal ◽

10.1042/bj1940611 ◽

1981 ◽

Vol 194 (2) ◽

pp. 611-620 ◽

Cited By ~ 36

Author(s):

M E Newman ◽

J Patel ◽

H McIlwain

Keyword(s):

Binding Site ◽

Adenosine Deaminase ◽

Binding Capacity ◽

Receptor Site ◽

Mammalian Brain ◽

Affinity Binding ◽

Temperature Dependent ◽

High Affinity ◽

High Affinity Site ◽

Uptake Process

1. A high-affinity adenosine-binding site with Kd(adenosine) 0.5-1.3 microM was demonstrated in particulate and synaptosomal fractions isolated from the cerebral cortex of guinea pig, rat and ox. 2. Binding of [3H]adenosine to this site was inhibited by theophylline and by 2-chloroadenosine, but not by four other adenosine analogues. 3. Endogenous adenosine, found to be present in some preparations at approx. 1 pmol/mg of protein, diminished the binding capacity of the preparations for [3H]adenosine. 4. Addition of the adenosine deaminase inhibitor erythro-9-[1-(1-hydroxyethyl)heptyl]-adenine revealed the presence of a second lower affinity binding site with Kd (adenosine) 5-9 microM and a higher maximal adenosine-binding capacity. The inhibitor partially blocked binding to the high-affinity site in preparations from which adenosine deaminase had been removed by washing. 5. To preparations of particulate fractions maintained under iso-osmotic conditions, adenosine attachment was non-saturable and temperature-dependent, indicating the existence of an active uptake process. 6. The location and binding constant of the high-affinity adenosine-binding site suggest that it corresponds to the receptor site for adenosine-activated adenylate cyclase.

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Modulation of skeletal muscle Ca2(+)-release channel activity by sphingosine

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.5.c1465 ◽

1997 ◽

Vol 272 (5) ◽

pp. C1465-C1474 ◽

Cited By ~ 7

Author(s):

D. H. Needleman ◽

B. Aghdasi ◽

A. B. Seryshev ◽

G. J. Schroepfer ◽

S. L. Hamilton

Keyword(s):

Skeletal Muscle ◽

Lipid Bilayers ◽

Binding Protein ◽

Planar Lipid Bilayers ◽

Dependent Manner ◽

Carboxy Terminus ◽

Release Channel ◽

Proteolytic Fragment ◽

High Affinity ◽

High Affinity Site

The effect of D-erythro-C18-sphingosine (sphingosine) and related compounds on the Ca(2+)-release channel (ryanodine binding protein) was examined on rabbit skeletal muscle membranes, on the purified ryanodine binding protein, and on the channel reconstituted into planar lipid bilayers. Sphingosine inhibited [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes in a dose-dependent manner similar to published results (R. A. Sabbadini, R. Betto, A. Teresi, G. Fachechi-Cassano, and G. Salviati. J. Biol. Chem. 267: 15475-15484, 1992). The sphingolipid also inhibited [3H]ryanodine binding to the purified ryanodine binding protein. Our results demonstrate that the inhibition of [3H]ryanodine binding by sphingosine is due to an increased rate of dissociation of bound [3H]ryanodine from SR membranes and a decreased rate of association of [3H]ryanodine to the high-affinity site. Unlike other modulators of the Ca(2+)-release channel, sphingosine can remove bound [3H]ryanodine from the high-affinity site within minutes. Sphingosine increased the rate of dissociation of [3H]ryanodine bound to a solubilized proteolytic fragment derived from the carboxy terminus of the ryanodine binding protein (cleavage at Arg4475). Sphingosine also inhibited the activity of the Ca(2+)-release channel incorporated into planar lipid bilayers. Taken together, the data provide evidence for a direct effect of sphingosine on the Ca(2+)-release channel. Sphingosine is a noncompetitive inhibitor at the high-affinity ryanodine binding site, and it interacts with a site between Arg4475 and the carboxy terminus of the Ca(2+)-release channel.

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