Biomedical subjects
Publications and source records attributed to E Shaw.
This paper reports a rather remarkable phenomenon, which we have termed "infantilization." Young acaras, 7 to 17 days postspawning age and normally freely swimming, will resume their nesting phase when placed with foster parents' brooding embryos or wrigglers. The foster parents do not eat these alien young when the young assume a behavior pattern similar to that of their own brood, namely, nest-bound and quiescent.
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Thrombocytin, a platelet-activating enzyme from Bothrops atrox venom, has been purified to homogeneity by precipitation with sodium salicylate and chromatography on heparin--agarose. Thrombocytin is a single-chain glycoprotein with a molecular weight of 36 000 which contains 5.6% carbohydrate. It causes platelet aggregation, release of platelet serotonin, and activation of factor XIII. The most sensitive substrate for the amidolytic activity of thrombocytin was Tos-Gly-Pro-Arg-p-nitroanilide hydrochloride. The activity of thrombocytin on this substrate and on platelets was inhibited by diisopropyl fluorophosphate (DFP), soybean trypsin inhibitor, and several arginine chloromethyl ketones. Active site titration with nitrophenyl guanidinobenzoate demonstrated that approximately 86% of the preparation was in the active form. These experiments demonstrate the presence of serine and histidine in the active site of thrombocytin and suggest that thrombocytin is a classical serine protease with a platelet-activating activity similar to thrombin.
Pro-Gly-ArgCH2Cl, a reagent corresponding to the C-terminal sequence generated in plasminogen on activation by urokinase (EC 3.4.99.26) and probably by other plasminogen activators, was prepared. Pro-Gly-ArgCH2Cl was effective in the inactivation of urokinase at the 10(-6) M level (Ki 68 micrometers and k2 0.47 min-1). In contrast, only a slow inactivation was obtained by 10(-2) M N-tosyllysine chloromethyl ketone. Glu-Gly-ArgCH2Cl, N,N-dimethylaminonaphthalene-5-sulfonyl-Glu-Gly-ArgCH2Cl, and Ac-Gly-Gly-ArgCH2Cl were more reactive than Pro-Gly-ArgCH2Cl against urokinase by factors of 25, 6, and 3, respectively. The effectiveness of arginine chloromethyl ketones as affinity labels is highly dependent on binding in the S2 and S3 sites, thus sequence variations in the reagents exhibited differences in reactivity of up to four orders of magnitude. The most effective reagents had Gly in P2. Ac-Gly-Gly-ArgCH2Cl inactivates urokinase 50 times more rapidly than it does plasmin, thus providing a means of distinguishing the activity of plasmin from its activating protease whereas urokinase is almost inert to Ala-Phe-LysCH2Cl, a reagent which inactivates plasmin at the 10(-7) M level.
The binding specificities of human urinary urokinase (EC 3.4.99.26) and HeLa cell plasminogen activator were studied using peptidyl chloromethyl ketone inhibitors. A 125I-labeled fibrin assay has been developed to yield kinetic information. Reagents of the sequence X-Gly-ArgCH2Cl were the most effective. The susceptibility of the HeLa cell plasminogen activator differed from that of urokinase in several respects indicating the utility of this type of inhibitor in distinguishing between proteases of this specificity.
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The synthesis of factor VIII and other coagulation factors has been studied using an isolated, perfused rat liver. Synthetic function of the liver was validated by adding [35S]L-methionine to the perfusion medium and performing two-dimensional crossed immunoelectrophoresis and autoradiography on samples obtained during perfusion. Progressive incorporation of radioactivity into plasma proteins was demonstrated. This was inhibited by cycloheximide. Coagulation factor assays demonstrated synthesis of factors II, IX and X and of factor V and procoagulant factor VIII (VIIIC). Synthesis of factor VIII related antigen (VIIIRAg), measured in an immunoradiometric assay, was not significantly demonstrated. Addition of warfarin to the perfusion medium inhibited the synthesis of factors II, IX and X but not of factors V and VIII. Cycloheximide completely inhibited synthesis of all coagulation factors but actinomycin acted only after a latent period. Reticuloendothelial cell blockade was attempted by adding ethionine to the perfusion medium or by administration of Indian ink to the donor animals prior to removal of the livers. In these instances synthesis of factor V and factor VIIIC was inhibited but not that of factors II, IX and X. The results confirmed the functional capacity of the isolated liver for synthesizing proteins and the vitamin K dependent coagulation factors, and suggested similar kinetic features for the synthesis of factors V and VIIIC. Failure to detect significant VIIIRAg synthesis in these experiments is consistent with the hypothesis that this protein is released by vascular endothelial cells throughout the body and is activated or joined to VIIIC or stimulates its production in the liver.
Synthetic procedures have been developed for the preparation of peptides of arginine chloromethyl ketone and applied in the preparation of affinity labels which correspond to the -Pro-Phe-Arg- C terminus of bradykinin, a physiological cleavage site of kallikrein in kininogen. Two such reagents, Ala-Phe-ArgCH2C1 and Pro-Phe-ArgCH2C1, proved to be highly effective as well as selective affinity labels for human plasma kallikrein. For example, Pro-Phe-ArgCH2C1 inactivates plasma kallikrein 50% in 24 min at a concentration of 2 x 10(-8)M, while other trypsin-like proteases are less susceptible in inactivation than kallikrein, differing by a factor of 48 for plasmin and factors of 10(2)-10(5) for factor Xa, thrombin, and urokinase. The affinity of human plasma kallikrein for Ala-Phe-ArgCH2C1 (Ki = 0.078 micron) is about 60 times that for Ala-Phe-LysCH2C1(Ki = 4.9 micron), whereas human plasmin exhibits about the same affinity for the former affinity label (Ki = 1.3 micron) as for the latter (Ki = 0.83 micron). The rate constants for the irreversible step of the affinity labeling reaction, k2, are similar for affinity labels tested with the individual proteases: 0.35 min-1 for plasma kallikrein and 0.18 min-1 for plasmin.
Attempts to achieve selective inactivation of serine proteases of closely related specificity (trypsin-like) by aryl sulfonylation have been extended. Nitrophenyl esters of benzenesulfonic acid and phenylmethanesulfonic acid containing various positively charged groups have been synthesized and examined as inactivators of trypsin, thrombin, plasmin, plasma kallikrein, and urokinase. Examples of selective inactivation by isothiouronium derivatives were found and attributed to differences among these enzymes in geometry and flexibility of the primary specificity sites.
A survey of the reactivity of 16 peptidyl-argininyl-chloromethanes with boar acrosin indicated that these compounds as a general group of reagents were highly effective in the inactivation of acrosin since at least half of the reagents tested rapidly inactivated this protease at a concentration of 0.10 micrometer or lower. For example, Dns-Glu-Gly-ArgCH2Cl inactivates acrosin by 50% in 1.8 min at a concentration of 75 nM, whereas in contrast, a 14000-fold higher concentration of Nalpha-tosyllysyl-chloromethane is required to obtain an equivalent rate of inactivation. A comparison of the reactivity of acrosin and trypsin with the peptides of arginyl-chloromethane containing different substituents in the P2 and P3 positions suggests that the secondary binding sites of these two proteases are very similar. Reagents with homoarginine, lysine and D-arginine in the P1 position have also been prepared and evaluated, but these were considerably less effective than the corresponding arginyl-chloromethanes in the inactivation of both acrosin and trypsin.
The diazomethyl ketones of z-Phe-Phe inactivate papain by a stoichiometric reaction at the active-center thiol. Since the reagents are stable in mercaptoethanol, their reaction with papain is judged to be the result of complex formation characteristic of affinity-labeling reagents. The diazomethyl ketones react by a mechanism different from that of chloromethyl ketones, since the pH dependence of their inactivation of papain is different, the rate increasing with decreasing pH. This relationship has been observed in other cases, such as in the reaction of azaserine with glutamine amidotransferases [Buchanan, J. M. (1973), Adv. Enzmol. Relat. Areas Mol. Biol. 39, 91], and is interpreted as an indication of reaction with a thiol group in its protonated form.
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A method for estimation of amoxycillin in plasma is described, based on the formation of a fluorescent derivative which is measured in the presence of 2-methoxyethanol. Drugs commonly administered together with amoxycillin do not appear to interfere with the assay. Data on the sensitivity, accuracy and linearity of the assay are presented together with a comparison with the microbiological assay.
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Affinity-labeling reagents are useful for the inactivation of proteases in vivo but are apparently limited in application by the possibility of side reactions. In addition, increased specificity would be desirable. Substrate-derived chloromethyl ketones, one class of protease inhibitors, have been examined with the hope that replacement of the departing group halogen by other substituents might lead to improved inhibitor characteristics. Analogues of Z-Phe-Ch2X were synthesized in which X is a sulfonate or carboxylate substituent and examined as inactivators of chymotrypsin. The sulfonate esters were found to be more reactive than the previously studied halogen derivatives.