Plasmin-like enzymes

Study of Activity of Plasmin-Like Enzymes in Tear... 

Activity of plasmin-like enzymes was measured with synthetic peptide substrate -Tos-Gly-Pro-Lys-p-nitroanilide ("Serva", Germany). Tear samples (5-25 (jl ) were placed into microliter plate wells. 150 fil of 0.05 M K-phosphate (pH 7.9) and 40 /il of substrate (2.4 mg/ml) were added in plate well for reaction initiation. Microtiter plate was diermostated at 250 °C. Optical density changes were measured using Microrider (Uniscan 11, Labsystems, Finland) at 405 nm. Measurements were made for 3-4 times during 5 hours. Plasmin-like activity of tear was calculated as tangent of slop of line in coordinates optical density via time. For accurate calculation we used a special software -Enzfitter (version 1.05 - EGA). We determined arbitrary unit of plasmin-like activity of tear as change of 1.0 unit of optical density per minutes per 1 ml of tear. 

Activity of plasmin-like enzymes of tear of healthy patients with intact cornea was 50.72 + 7.73 AU. At the first day of observation the activity of lacrimal plasmin-like enzymes of patients with corneal epithelial defects in the control group increased more than 4 times and was 234.5 19.0 AU. These data did not significantly differed from the data obtained for the patients from the first experimental group (249.0 21.5 AU) (table 1). On the contrary, the activity of lacrimal plasmin-like enzymes in patients who had small and strong drinks during the time of the present study (second group) increased almost 1.5 time and was 352.6 24.2 AU (p < 0.01). 

Activity of plasmin-like enzymes of the tear of patients with corneal epithelial... 

At the second day of observation use of the protease inhibitor contrycal allowed to reduce activity of plasmin-like enzymes to 168.5 18.6 AU in the control group and to 153.7 19.6 AU in the first experimental group. This activity was also reduced to 294.2 18.0 AU in the second experimental group, but the decrease of activity was not so significant as in the other groups. 

The results of our study confirm that all patients with comeal epithelial defects have increased activity of plasmin-like enzymes of tear, which was almost 1.5 times higher in the second group dian in the control one. 

It is well known that the specificity of an enzyme such as thrombin and plasmin is very close to that of trypsin. In this respect, inverse substrates for trypsin also are expected to be susceptible to the catalysis by these enzymes. In the kinetic analysis of trypsin-like enzymes toward p-amidinophenyl esters, it was found that the inverse concept is also applicable to thrombin, plasmin, urokinase, kallikrein, and trypsins from various origins 74 - 75). These enzymes are not distinctively different from bovine-... 

The active site structure of trypsin-like enzymes is considered to be very similar to that of bovine trypsin, yet little is known about them. Refinement of these structures is important also for the purpose of designing physiologically active substances. With a view to comparing the spatial requirements of active sites of these enzymes, dissociation constants of the acyl enzyme-ligand complex, K-, which were defined before, were successfully analyzed By taking advantage of inverse substrates which have an unlimited choice of the acyl component, development of stable acyl enzymes could be possible. These transient inhibitors for trypsin-like enzymes could be candidates for drugs. In this respect, the determination of the deacylation rate constants for the plasmin- and thrombin-catalyzed hydrolyses of various esters were undertaken 77). 

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. 

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. 

The leupeptins are Inhibitors of plasmin, a trypsin-like enzyme. Leupeptlns contain arginlnyl residues at their terminal carbon, and inhibit enzymes which cleave at the carboxyl side of basic amino acids such as arginine or lysine.50 The structure of the most active leupeptin mixture is propionyl-L-Leu-L-Leu-Argininal and acetyl-L-Leu-L-Leu-Arglnlnal in a 3 1 ratio.64-66... 

D. Thrombin-Like Enzymes. Ancrod (arvin), a very specific snake venom component, has been investigated extensively for its anticoagulant action. Ancrod is a very specific protease whose action has some similarity to that of thrombin and occurs in the terminal sequence of a complex blood coagulation mechanism. Ancrod hydrolyzes only the Aa chain of fibrinogen and produces a polymer of the type (a Bp y)n rather than the (aPy)n type normal fibrin clot. The microclot produced by ancrod from fibrinogen is readily hydrolyzed by plasmin that was activated from tissue plasminogen. This results in a defibrination effect. This property is extensively used in the treatment of a patient who has suffered from myocardial infarction. 

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). 

Studies by many workers have revealed two peptide sequences which are very selective for thrombin -Phe-Val-Arg- and -X-Pro-Arg- (C5, S24, S26). As can be seen in Table 3, these two sequences have been widely used to design the various synthetic substrates to detect thrombin. This measurement can be done directly by incubating this enzyme with the appropriate substrate. Latallo has recommended adding an agent like aprotinin or soybean trypsin inhibitor to prevent attack by kallikrein, Factor Xa, or plasmin on these thrombin substrates (L5). The use of the original substrate, S-2160, has sharply declined due to its lower sensitivity, specificity, and solubility when compared to substrates like S-2238 and Chromozyn TH. Today,... 

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