The proteolytic action of Arvin on human fibrinogen

1. Human fibrinogen was subjected to proteolysis by enzyme preparations (clinical Arvin and IRC-50 Arvin) from the venom of Agkistrodon rhodostoma. 2. IRC-50 Arvin releases three peptides from fibrinogen, and these were identified as fibrinopeptides AP, AY and A. 3. The less purified ;clinical' Arvin releases, in addition to fibrinopeptides AP, AY and A, small amounts of two heptapeptides derived from fibrinopeptides AP and A, probably because it contains another enzyme as well as Arvin. 4. No fibrinopeptide B is released by either Arvin preparation. 5. Thus, although Arvin is known to differ from ;reptilase' from Bothrops jararaca in that it does not activate the enzyme that cross-links fibrin (fibrin-stabilizing factor), it is identical with reptilase with respect to the peptides that it liberates from fibrinogen.     

The proteolytic action of bacterial proteases from Clostridium histolyticum on bovine fibrinogen.

Bacterial proteases from Clostridium histolyticum bring about the polymerization of fibrinogen into structured fibres, the period being 9 nm when the ionic strength (I) of the solution is between 0.1 and 0.2. At I=0.3 no polymerization occurs, but the successive actions of bacterial proteases and thrombin induce polymerization, the fibres obtained showing a periodic structure: the major period is 23 nm with two minor striations. The striation pattern is different from that obtained with fibrin which shows a major period of 23 nm with three minor striations. The degree of degradation of fibrinogen monitored by disc gel electrophoresis shows that the Aalpha polypeptide chain (mol. wt.=68 000) is degraded into Aalpha1 (mol. wt.=32 000) and Aalpha2 (mol. wt.=29 000), whilst the mobility of Bbeta increases and gamma remains unchanged; the molecular weight is estimated between 272 000 and 269 000. These results emphasize that the charge distribution differences which are compatible with the loss of different portions of the molecule, lead to variations in fibre structures.     

The action of proteolytic enzymes on chloroplast thylakoid membranes

Envelope- and stroma-free thylakoid membranes of Vicia faba chloroplasts were incubated with trypsin or pronase for several hours. The indigestible residue was analyzed by polyacrylamide gel electrophoresis. Trypsinization resulted in a complete digestion of all proteins with the exception of the pigment-protein complexes as well as a polypeptide not yet characterized. Yet, as compared with untreated material, Complex II was found to have higher electrophoretic mobility. Electron-microscopic studies illustrate that the indigestible residue still has a preserved membrane structure. Disintegration of the thylakoid membranes by sodium dodecyl sulfate followed by trypsinization also resulted in the two complexes while all the other proteins were found to be digested. However, after removal of the lipids the protein moieties of the complexes proved to be easily digestible. From these results it is concluded that pigment-protein interaction may be an important factor in maintaining a conformation rather resistant to perturbants and proteases. In contrast to trypsin, pronase completely digested the polypeptides of the thylakoid membranes including the protein moieties of the pigment-protein complexes leaving an amorphous lipid mass. The results support the assumption that the complexes are necessary to maintain the membrane structure.     

The action of proteolytic enzymes on the glycoprotein from pig gastric mucus.

A glycoprotein of mol.wt. 2x10(6) was isolated in homogeneous form from pig gastric mucus by isopycnic centrifugation in CsCl but without enzymic digestion or reductive cleavage of disulphide bonds. Digestion of the purified glycoprotein with trypsin, pepsin or Pronase resulted in the formation of glycoprotein subunits, of mol.wt. 5.2x10(5)-5.8x10(5), one-quarter that of the undigested glycoprotein. The glycoprotein subunits were isolated by gel filtration and shown to contain all the carbohydrate present in the undigested glycoprotein, but 18.6-25.6% of the total amino acids originally present were lost on digestion. The relative amount of threonine, serine and proline had increased from 41% (w/w) in the undigested glycoprotein to 61-67% of the total amino acids in the glycoprotein subunits after digestion. The results support the previously proposed structure for the glycoprotein, namely that of four subunits joined by disulphide bridges. These results show the presence of two distinct regions in the glycoprotein molecule, one rich in threonine, serine and proline, which is glycosylated and resistant to proteolyis, whereas the other, with an amino acid composition more characteristic of a globular protein, is not glycosylated and is susceptible to proteolysis. In addition, the region that is susceptible to proteolysis contains the disulphide bridges which join the glycoprotein subunits together to form the gastric glycoprotein.     

Mechanism of action of thrombin on fibrinogen. Reaction of the N-terminal CNBr fragment from the Aalpha chain of human fibrinogen with bovine thrombin

The 51-residue N-terminal cyanogen bromide fragment from the Aα chain of human fibrinogen was isolated, and the Michaelis-Menten constants, K_m and k_cat, for its hydrolysis by bovine thrombin were determined. The measured values of K_m and k_cat are 4.7 × 10^?5m and 4.8 × 10^?10m [(NIH U/liter) sec]^?1, respectively. Since these values are similar to those for fibrinogen, it appears that the N-terminal CNBr fragment contains all amino acid residues whose interactions with thrombin account for the high specificity of this enzyme for fibrinogen.