Details

Autor(en) / Beteiligte

• Soler-Ferran, Dulce F

Titel

1. Purification and characterization of atrial dipeptidyl carboxyhydrolase. 2. Development of heparin-binding peptides

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

1991

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Atrial dipeptidyl carboxyhydrolase is a zinc-metallo protease that cleaves C-terminal dipeptides from short chain peptide hormones and synthetic peptides. The enzyme catalyzes the conversion of atriopeptin II, one atrial peptide, to another, atriopeptin I, by removal of Phe-Arg. The enzyme also acts as a tripeptidyl carboxyhydrolase with atriopeptin III as substrate. Substrate specificity studies revealed the amino acid preferences at the P$\sb1$ and P$\sb1\sp\prime$ positions in the substrate. The enzyme was purified $\approx$10,000 fold (specific activity 6 unit/mg). This enzyme is a glycoprotein of molecular weight 250,000. During purification of atrial dipeptidyl carboxyhydrolase, a bacterial enzyme possessing the same catalytic activity was discovered. This enzyme is also a zinc-metallo proteinase, and shares some kinetic properties with atrial dipeptidyl carboxyhydrolase. The bacterial enzyme is $\approx$80,000 in molecular weight, has a different inhibitor profile, and displays different substrate specificities. The study of heparin-protein interactions is important for an understanding of the regulation of protein function by heparin. von Willebrand factor is a plasma glycoprotein which plays an important role in the regulation of hemostasis through its interaction with platelets and exposed subendothelium. von Willebrand factor interacts with heparin which inhibits its interaction with platelets. To study the interaction of heparin with vWF, a consensus peptide which bound heparin with high affinity was designed and synthesized. The motif of the consensus peptide was used to identify a potential heparin binding domain in vWF. The heparin binding domain in vWF was mapped to a peptide of 24 amino acids corresponding to Tyr$\sp{565}$-Val$\sp{587}$ which showed high binding affinity for heparin and successfully competed with vWF for binding heparin. This peptide was used for fractionation of heparins which showed high affinity for vWF. A peptide designed to be $\alpha$-helix in character was synthesized to study the sequence and structural requirements necessary for binding heparin. The interaction of heparin with the helix peptide was monitored by circular dichroism spectrometry; heparin increases the helix content of the peptide and also increases the stability of the helix-peptide. Thus, the helix peptide can be used as a framework to study protein-heparin interactions mediated by helix structures.