Plasmin labelled

Fig. 2. Schematic diagram of the three pairs of polypeptide chains of fibrinogen. The Aa, B/3, and 7 chains are represented by bars with lengths proportional to the number of amino acid residues in each chain and the N- and C-terminal ends of the chains are labeled. The coiled-coil regions are indicated by the diagonally striped boxes, while the intra- and interchain disulfide bonds are indicated by solid lines. Carbohydrate attachment sites are labeled with CHO, while thrombin and major plasmin cleavage sites are indicated by T and P, respectively. (Adapted from Fig. 12-1 of Hantgan et al., 2000.)...

Fio. 2. Plasmin and uPA-catalyzed cleavage of suPAR(I-III). (A) 35S-labeled recombinant suPAR(I-III) was purified on an immunoaffinity column with the domain I-specific mAb R3 immobilized [86], Lane 1 is 10 nM suPAR(I-III), lane 2 is 10 nM suPAR(I-III) incubated for 2 hours at 37 °C with 50 nM plasmin (pli), lane 3 is 10-nM suPAR(I-III) incubated for 20 hours at 37°C with 500-nM uPA, and lane 4 is 35S-labeled domain I purified from cell culture media on an immunoaffinity column with the domain I-specific mAb R9 immobilized, after removing suPAR (I—III) from the media by immunoaffinity chromatography, employing the domain Ill-specific mAb R2. (B) Ten nM suPAR(I-III) was preincubated with 67 nM of the indicated mAbs for 2 hours at 37°C prior to addition of either 50 nM plasmin (lanes 2,3,4, and 6) or 500 nM uPA (lane 5) and continued incubation for 2 or 20 hours at 37 °C. Lane 1 is suPAR(I-III) alone, lane 2 is suPAR(I-III) incubated with plasmin only, lane 3 shows preincubation with a subtype control mAb, lanes 4 and 5 show preincubation with mAb R3, lane 6 shows preincubation with the domain Ill-specific mAb R4. The radioactive samples have been separated by SDS-PAGE prior to autoradiography of the dried gel. 

The results on the hydrolysis of partially methylated /3-casein by plasmin indicate that proteins radiomethylated to a low level can serve as substrates for trypsin-like enzymes and probably for proteinases in general. Because it is likely that methylation will interfere with enzymatic attack at lysine residues, the complete hydrolysis of /3-casein probably would not be possible. Studies on mastitic milk demonstrate the usefulness of 14C-methyl proteins for qualitative examination of protein hydrolysis in complex multiprotein systems where resolution and characterization of individual protein fragments is difficult. The requirements in such studies are the availability of pure samples of the proteins under investigation and a suitable technique for separating the radio-labeled protein from hydrolytic products. 

Heparin has been fluorescently labeUed in a manner that does not alter the functional properties of the polysaccharide. The labelled polysaccharide has been used in conjunction with fluorescence polarization spectroscopy to monitor the binding of heparin to the L-serine proteases thrombin, factor XIa, factor Xa, and plasmin. The stoicheiometry and dissociation constants of the interactions have been measured. The kinetics of inactivation of the four proteases by anti-thrombin as a function of heparin concentration have also been measured. Evidence shows that the direct binding of heparin to anti-thrombin is probably responsible for the polysaccharide-dependent acceleration of hemostatic enzyme-inhibitor reactions. 

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