Plasmin hydrolysis

Transition state theory, 46,208 Transmission factor, 42,44-46,45 Triosephosphate isomerase, 210 Trypsin, 170. See also Trypsin enzyme family active site of, 181 activity of, steric effects on, 210 potential surfaces for, 180 Ser 195-His 57 proton transfer in, 146, 147 specificity of, 171 transition state of, 226 Trypsin enzyme family, catalysis of amide hydrolysis, 170-171. See also Chymotrypsin Elastase Thrombin Trypsin Plasmin Tryptophan, structure of, 110... 

Thrombin [EC 3.4.21.5], also known as fibrinogenase, catalyzes the hydrolysis of peptide bonds, exhibiting preferential cleavage for the Arg—Gly peptide bond. The enzyme, a member of the peptidase family SI, activates fibrinogen to fibrin and releases fibrinopeptide A and B. Thrombin, formed from prothrombin, is more selective in peptide hydrolysis than trypsin or plasmin. 

Streptokinase Trypsin Uiicase Urokinase Plasminogen- plasmin Protein hydrolysis Urate+02->allantoin Plasminogen->plasmm Blood clots Inflammation Gout Blood clots... 

Hydrolysis of primary caseins by plasmin. In 1969, Groves and coworkers showed that the y-casein fraction, as isolated by Hipp et al., is very heterogeneous, containing at least four distinct proteins y-casein, temperature-sensitive casein (TS, which is soluble in the cold but precipitates above 20°C), R-casein and S-casein. These four proteins were shown to be C-terminal fragments of /3-casein. In 1976, the nomenclature of the y-casein group was revised, as shown in Figure 4.7 and Table 4.3. 

Proteinase (plasmin) Hydrolysis of peptide bonds, particularly in -casein Reduced storage stability of UHT products cheese ripening... 

The other major casein in cheese is /3-casein, but it is generally not hydrolyzed by rennet in low-pH cheeses. Alkaline milk protease (plas-min) plays the major role in the hydrolysis of /3-casein (Richardson and Pearce 1981). The plasmin level in cheese is related to the pH of the curd at whey drainage, since plasmin dissociates from casein micelles as the pH is decreased. Richardson and Pearce (1981) found two or three times more plasmin activity in Swiss cheese than in Cheddar cheese. Swiss cheese curds are drained at pH 6.4 or higher, while Cheddar cheese curds are drained at pH 6.3 or lower. Proteolysis of /3-casein is significantly inhibited by 5% sodium chloride. The inhibitory influence of sodium chloride is most likely due to alteration of /3-casein or a reduction in the attractive forces between enzyme and substrate (Fox and Walley 1971). 

Fig. 8. Kinetics of unstabilized fibrin hydrolysis with complexes of immobilized heparin with fibrinogen (1), thrombin (2), fibrinolysin (plasmin) (3), and serum albumin (4),06)...

Plasmin Hydrolysis of /3-Casein. Studies on the susceptibility of partially methylated /3-casein to cleavage by trypsin-like enzymes were carried out using the enzyme porcine plasmin. In preliminary investigations, we confirmed that the y-caseins produced by plasmin hydrolysis of native /3-casein were identical with those occurring naturally (28). These are designated yi-, y2-, and y3-casein according to the nomenclature recommendations of Whitney et al. (29) and correspond to residues 29-109, 106-209, and 198-209 of -casein. Presumably the proteose peptone products of plasmin hydrolysis are identical with their natural counterparts, but the latter were not available for comparison. 

Plasmin Hydrolysis of 14C-Methyl-/3-Casein. On the basis of a study by Ottesen and Svensson (2), which described the relative amounts of mono- and dimethyllysinyl derivatives produced by reductive methyla-... 

P-Casein (13 mg) containing 0.036 fid M-P-C was subjected to plasmin hydrolysis for 45 min and the reaction mixture was dissolved in 7 mL column buffer (5mM Tris—3mM NaCl—urea, pH 8.55) together with 100 mg whole casein that had been alkylated with iodoacetamide. The sample solution was applied to a column (1.6 X 50 cm) of DEAE-cellulose equilibrated with column buffer. Elution was with a NaCl gradient (3.0-155mM) in column buffer (gradient volume, 1.0 L) 5.0 mL fractions were collected. Under the conditions used as as-caseins remained adsorbed to the column K-caseins were eluted in Fractions 35-56 Am measurement (----------) radioactivity (--) (28). 

Figure 7. PAGE of reaction mixture after plasmin hydrolysis of B-casein for 0, 20, 30, 40, and 70 min (Slots 1-5) and Fractions 1 ana 2 from hydroxyapatite chromatography of hydrolysis products after plasmin treatment of fi-casein for 70 min (Slots 6 and 7). Fraction 2 contains a further phosphopeptide that is not visible in Slot 7 but appears on disc gels in the expected position for proteose peptone component 8F (cf. Ref. 32) (28).

Rate of Plasmin Hydrolysis. It may be concluded from the above electrophoretic and chromatographic data that partial methylation of a protein does not interfere with the site specificity of plasmin hydrolysis or result in the production of artifactual hydrolytic products. It remained to show what effect methylation had on the rate of hydrolysis by plasmin. M-/3-C was diluted approximately 300-fold with unlabeled protein and subjected to plasmin hydrolysis. The reaction mixture was sampled at various intervals up to 70 min, by which time most of the /3-casein was transformed (see Figure 7, Slots 1-5). The extent of transfer of radioactivity to the reaction products was examined to determine... 

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. 

This work was supported in part by Ross Laboratories (Columbus, OH), the College of Agricultural and Life Sciences, University of Wisconsin—Madison, the Cooperative State Research Service, USD A, and by the Irish Agricultural Institute. T. Richardson was a Fulbright-Hays Scholar at the Agriculural Institute in Fermoy, County Cork, Ireland, where the hydrolysis of methylated /3-casein by plasmin was studied. The authors are grateful for helpful discussions with L. K. Creamer. 

Bradykinin and kallidin ate potent vasodilators and hypotensive agents that have different peptide structures bradykinin is a nonapeptide, whereas kallidin is a decapepttde. Kdlidin is ly.syl-bradykinin that is. it has an additional lysine at the NH2 terminus of the chain. Tliese two compounds arc made available from kininogen. a hlood globulin, on hydrolysis. Trypsin, plasmin, or the proteases of certain snake venoms can catalyze the hydrolysis of kininogen. 

The normal tissue lysis activators are proteins. One such activator from the kidney is urokinase. This enzyme has not been found systemically, but only in the kidney and urine. It directly activates plasminogen to plasmin by hydrolysis of a small peptide from the plasminogen molecule. Other known enzymatic activators are streptokinase and brinase, which have been discussed elsewhere.82>83 Urokinase can be isolated from urine in small amounts. This has made it a very expensive material. Abbott Laboratories has developed a tissue culture technique in which kidney cells are grown under exacting conditions and urokinase is isolated.84 Recently Abbott Laboratories made a request to the FDA for a license to sell this form of urokinase called Abbokinase . This process should increase the availability of urokinase and decrease its cost. 

Caessens, PJ.W.R. et al., P-Lactoglobulin hydrolysis. 1. Peptide composition and functional properties of hydrolysates obtained by the action of plasmin, trypsin, and Staphylococcus aureus V9 protease, J. Agric. Food Chem., 47, 2973, 1999. 

De Groot et al. used the same approach to prepare prodmgs of paclitaxel for activation by plasmin.69 However, most of the prepared prodmgs were either not stable or resistant to the hydrolysis by plasmin. An alternative approach using a p-amino-benzyl alcohol group as a spacer appeared to be successful. Prodrug 36 yielded free paclitaxel (36a) upon incubation with plasmin (Scheme 18). Further, prodmg 36 showed a dramatic decrease in cytotoxicity in seven human tumor cell lines in... 

Paclitaxel 2 -carbamates and 2 -carbonates were prepared to fulfill the requirements of an ideal prodrug, that is, low cytotoxicity for healthy tissue, high stability against unspecific enzymes, and fast hydrolysis by the tumor-associated enzyme. These water-soluble prodrugs are specifically activated by plasmin in the tumor cells and show, on average, a decrease in cytotoxicity of more than 8,000-fold in comparison with the parent drug. ... 

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