DOMAIN OF BINDING ACTIVITY WITH PLASMIN KRINGLE IN SYNTHESIZED C-TERMINAL PEPTIDES , OF α2-PLASMIN INHIBITOR

The inhibitory reaction between plasmin and α2-plasmin inhibitor (α2-PI) proceeds with two steps, a very fast reversible reaction followed by a slower irreversible transition. The first step is dependent on the interaction between lysine binding site (LBS) of plasmin and the corresponding complementary site of α2-PI (kringle binding site(KBS)). It has been reported that KBS is located in a C-terminal tryptic fragment (T-11; J. Biochem. 99, 1699 (1986)).In order to investigate which amino acid residues of T-ll play important roles in binding of plasmin kringle, we tested inhibitory activity of synthesized peptides on the apparent rate constant in the reaction between α2-PI and plasmin. 50% inhibition concentrations of T-ll, peptide I, II, III and IV were 7, 18, 13, 35 and 250pM respectively, indicating that Leu9-Lysl0 is an important part for binding of T-ll to LBS. Peptide III lost its activity by depletion or amidation of the C-terminal lysine residue.In the system consisted of α2-PI and miniplasmin which lacked kringle 1-4, peptide I did not inhibit the interaction between them. Furthermore, peptide II competitively inhibited the binding of tranexamic acid to kringle 1-3 (Ki 0.85μM).These findings suggest that the C-terminal part is involved in the high affinity binding of α2-PI to plasmin kringle and that LyslO in T-ll and C-terminal carboxyl residue play crucial roles in binding to LBS of kringle.

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Localisation of a high affinity binding site for coat protein on the 3′-terminal part of RNA 4 of alfalfa mosaic virus

FEBS Letters ◽

10.1016/0014-5793(80)81279-8 ◽

1980 ◽

Vol 121 (1) ◽

pp. 123-126 ◽

Cited By ~ 5

Author(s):

Karel Stoker ◽

Ellen C. Koper-Zwarthoff ◽

John F. Bol ◽

E.M.J. Jaspars

Keyword(s):

Coat Protein ◽

Binding Site ◽

Mosaic Virus ◽

Alfalfa Mosaic Virus ◽

Affinity Binding ◽

Terminal Part ◽

High Affinity Binding ◽

High Affinity ◽

High Affinity Binding Site

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An AU-Rich Sequence Element (UUUN[A/U]U) Downstream of the Edited C in Apolipoprotein B mRNA Is a High-Affinity Binding Site for Apobec-1: Binding of Apobec-1 to This Motif in the 3′ Untranslated Region of c-myc Increases mRNA Stability

Molecular and Cellular Biology ◽

10.1128/mcb.20.6.1982-1992.2000 ◽

2000 ◽

Vol 20 (6) ◽

pp. 1982-1992 ◽

Cited By ~ 67

Author(s):

Shrikant Anant ◽

Nicholas O. Davidson

Keyword(s):

Binding Site ◽

Apolipoprotein B ◽

Half Life ◽

Rna Binding ◽

Cytidine Deaminase ◽

Binding Activity ◽

Affinity Binding ◽

High Affinity Binding ◽

High Affinity ◽

High Affinity Binding Site

ABSTRACT Apobec-1, the catalytic subunit of the mammalian apolipoprotein B (apoB) mRNA-editing enzyme, is a cytidine deaminase with RNA binding activity for AU-rich sequences. This RNA binding activity is required for Apobec-1 to mediate C-to-U RNA editing. Filter binding assays, using immobilized Apobec-1, demonstrate saturable binding to a 105-nt apoB RNA with a Kd of ∼435 nM. A series of AU-rich templates was used to identify a high-affinity (∼50 nM) binding site of consensus sequence UUUN[A/U]U, with multiple copies of this sequence constituting the high-affinity binding site. In order to determine whether this consensus site could be functionally demonstrated from within an apoB RNA, circular-permutation analysis was performed, revealing one major (UUUGAU) and one minor (UU) site located 3 and 16 nucleotides, respectively, downstream of the edited base. Secondary-structure predictions reveal a stem-loop flanking the edited base with Apobec-1 binding to the consensus site(s) at an open loop. A similar consensus (AUUUA) is present in the 3′ untranslated regions of several mRNAs, including that of c-myc, that are known to undergo rapid degradation. In this context, it is presumed that the consensus motif acts as a destabilizing element. As an independent test of the ability of Apobec-1 to bind to this sequence, F442A cells were transfected with Apobec-1 and the half-life of c-myc mRNA was determined following actinomycin D treatment. These studies demonstrated an increase in the half-life of c-myc mRNA from 90 to 240 min in control versus Apobec-1-expressing cells. Apobec-1 expression mutants, in which RNA binding activity is eliminated, failed to alter c-myc mRNA turnover. Taken together, the data establish a consensus binding site for Apobec-1 embedded in proximity to the edited base in apoB RNA. Binding to this site in other target RNAs raises the possibility that Apobec-1 may be involved in other aspects of RNA metabolism, independent of its role as an apoB RNA-specific cytidine deaminase.

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Lactoferrin-lipopolysaccharide interaction: involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide

Biochemical Journal ◽

10.1042/bj3120839 ◽

1995 ◽

Vol 312 (3) ◽

pp. 839-845 ◽

Cited By ~ 160

Author(s):

E Elass-Rochard ◽

A Roseanu ◽

D Legrand ◽

M Trif ◽

V Salmon ◽

...

Keyword(s):

Escherichia Coli ◽

Binding Site ◽

Binding Sites ◽

Affinity Binding ◽

High Affinity Binding ◽

Tryptic Fragment ◽

High Affinity ◽

Loop Region ◽

High Affinity Binding Site ◽

Lps Binding

The ability of lactoferrin (Lf), an iron-binding glycoprotein that is also called lactotransferrin, to bind lipopolysaccharide (LPS) may be relevant to some of its biological properties. A knowledge of the LPS-binding site on Lf may help to explain the mechanism of its involvement in host defence. Our report reveals the presence of two Escherichia coli 055B5 LPS-binding sites on human Lf (hLf): a high-affinity binding site (Kd 3.6 +/- 1 nM) and a low-affinity binding site (Kd 390 +/- 20 nM). Bovine Lf (bLf), which shares about 70% amino acid sequence identity with hLf, exhibits the same behaviour towards LPS. Like hLf, bLf also contains a low- and a high-affinity LPS-binding site. The Kd value (4.5 +/- 2 nM) corresponding to the high-affinity binding site is similar to that obtained for hLf. Different LPS-binding sites for human serum transferrin have been suggested, as this protein, which is known to bind bacterial endotoxin, produced only 12% inhibition of hLf-LPS interaction. Binding and competitive binding experiments performed with the N-tryptic fragment (residues 4-283), the C-tryptic fragment (residues 284-692) and the N2-glycopeptide (residues 91-255) isolated from hLf have demonstrated that the high-affinity binding site is located in the N-terminal domain I of hLf, and the low-affinity binding site is present in the C-terminal lobe. The inhibition of hLf-LPS interaction by a synthetic octadecapeptide corresponding to residues 20-37 of hLf and lactoferricin B (residues 17-41), a proteolytic fragment from bLf, revealed the importance of the 28-34 loop region of hLf and the homologous region of bLf for LPS binding. Direct evidence that this amino acid sequence is involved in the high-affinity binding to LPS was demonstrated by assays carried out with EGS-loop hLf, a recombinant hLf mutated at residues 28-34.

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