scholarly journals The Identity of the Nucleophile Substitution May Influence Metal Interactions with the Cleavage Site of the Minimal Hammerhead Ribozyme

Biochemistry ◽  
2009 ◽  
Vol 48 (44) ◽  
pp. 10654-10664 ◽  
Author(s):  
Edith M. Osborne ◽  
W. Luke Ward ◽  
Max Z. Ruehle ◽  
Victoria J. DeRose
Keyword(s):  
Cleavage Site ◽  
Hammerhead Ribozyme ◽  
Metal Interactions

Structural Variation Induced by Different Nucleotides at the Cleavage Site of the Hammerhead Ribozyme†

Biochemistry ◽  
1998 ◽  
Vol 37 (12) ◽  
pp. 4034-4044 ◽  
Author(s):  
Jean-Pierre Simorre ◽  
Pascale Legault ◽  
Narayan Baidya ◽  
Olke C. Uhlenbeck ◽  
Lara Maloney ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Structural Variation ◽  
Hammerhead Ribozyme

A Reappraisal, Based on31P NMR, of the Direct Coordination of a Metal Ion with the Phosphoryl Oxygen at the Cleavage Site of a Hammerhead Ribozyme

2002 ◽  
Vol 124 (28) ◽  
pp. 8230-8236 ◽  
Author(s):  
Ken-ichi Suzumura ◽  
Koichi Yoshinari ◽  
Yoshiyuki Tanaka ◽  
Yasuomi Takagi ◽  
Yasuhiro Kasai ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Metal Ion ◽  
Hammerhead Ribozyme ◽  
Phosphoryl Oxygen

Identification of the Hammerhead Ribozyme Metal Ion Binding Site Responsible for Rescue of the Deleterious Effect of a Cleavage Site Phosphorothioate†

Biochemistry ◽  
1999 ◽  
Vol 38 (43) ◽  
pp. 14363-14378 ◽  
Author(s):  
Shenglong Wang ◽  
Katrin Karbstein ◽  
Alessio Peracchi ◽  
Leonid Beigelman ◽  
Daniel Herschlag
Keyword(s):  
Binding Site ◽  
Cleavage Site ◽  
Metal Ion ◽  
Deleterious Effect ◽  
Hammerhead Ribozyme ◽  
Ion Binding ◽  
Metal Ion Binding

Properties of Chimeric (Tissue-Type/Urokinase-Type) Plasminogen Activators Obtained by Fusion at the Plasmin Cleavage Site

1993 ◽  
Vol 69 (05) ◽  
pp. 466-472 ◽  
Author(s):  
M Colucci ◽  
L G Cavallo ◽  
G Agnelli ◽  
A Mele ◽  
R Bürgi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Cleavage Site ◽  
Plasminogen Activators ◽  
Tissue Type ◽  
Low Molecular Weight ◽  
Single Chain ◽  
Type Plasminogen Activator ◽  
Kringle 2 ◽  
Fibrin Monomers

SummaryTwo hybrid plasminogen activators (K2tu-PA and FK2tu-PA), linking the kringle 2 domain or the finger plus the kringle 2 domains of tissue-type plasminogen activator (t-PA) to the catalytic domain of single-chain urokinase-type plasminogen activator (scu-PA) were studied. At variance with similar constructs previously reported, they were obtained by fusion of the t-PA and scu-PA derived portions at their plasmin cleavage site (between Arg275 of t-PA and Ile159 of scu-PA), thus eliminating from scu-PA the two peptide bonds (Glu143-Leu144 and Arg156-Phe157) that lead to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K2tu-PA and FK2tu-PA, as measured by fibrin plate were 2.5 × 106 and 1.0 × 106 t-PA equivalent units/mg, respectively. Activation of plasminogen by hybrid PAs was stimulated by both CNBr-digested fibrinogen (40- and 80-fold) and Des-A-fibrin monomers (6- and 12-fold). The relatively weak stimulation of chimeric PAs by minimally degraded fibrin monomers was consistent with their reduced fibrin binding capacity. Like scu-PA, the chimeric PAs, in the single-chain form, were insensitive to inhibition, as they retained full activity after prolonged incubation in plasma and did not interact with SDS-reactivated recombinant PAI-1. The concentration producing 50% lysis of blood clots in 3 h was 0.5 μg/ml for K2tu-PA and 1 μg/ml for FK2tu-PA, as compared to 0.5 μg/ml and >2 μg/ml for t-PA and scu-PA, respectively. Plasminogen and α2-antiplasmin consumption induced by the hybrid PAs in clot-free plasma was comparable to (K2tu-PA) or lower than (FK2tu-PA) that induced by either t-PA or scu-PA. When exposed to plasmin, the hybrids were completely converted into two-chain molecules with full enzymatic activity. At variance with u-PA, however, the two-chain recombinant activators still required fibrin for full expression of activity. These data indicate that the products of such “artificial” fusion behave like true chimeras without loss of biological activity. The insensitivity to thrombin inactivation and to the proteolytic cleavage leading to low molecular weight scu-PA might confer enhanced stability to the molecules, especially at thrombus level. Moreover, if the thrombolytic activity observed in vitro is maintained in vivo, the prolonged half life of these hybrids should result in higher plasma levels of activator and thus in more extensive and rapid lysis.

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