S L Gonias

Physical properties of human α2-macroglobulin following reaction with methylamine and trypsin

Abstract Circular dichroism spectroscopy, sedimentation velocity and ultraviolet difference spectroscopy were used to compare α 2 -macroglobulin, α 2 -macroglobulin-trypsin complex and α 2 -macroglobulin-methylamine complex. The circular dichroic spectrum of native α 2 -macroglobulin is significantly changed in shape and magnitude following reaction with either trypsin or methylamine. The spectra of α 2 -macroglobulin-trypsin and α 2 -macroglobulin-methylamine are, however, indistinguishable. The ultraviolet difference spectrum between α 2 -macroglobulin-methylamine and native α 2 -macroglobulin displays a tyrosine blue shift consistent with the exposure of several tyrosine residues to solvent. The conformational change which occurs in α 2 -macroglobulin during reaction with methylamine follows pseudo-first-order kinetics. T 1/2 was 10.5 min for the reaction with 200 mM methylamine at pH 8.0 and 45 min for the reaction with 50 mM methylamine, also at pH 8.0. Reaction of methylamine with α 2 -macroglobulin results in loss of trypsin-binding activity which appears to be a direct consequence of the conformational change induced by methylamine. A sedimentation coefficient (s o 20 ,w) of 20.5 was determined for α 2 -macroglobulin-methylamine compared to a value of 18.5 for unreacted α 2 -macrogiobulin. This increase in sedimentation velocity is attributed to a 10% decrease in α 2 -macroglobulin Stokes radius, α 2 -Macroglobulin-trypsin complex prepared by reaction of the protease ata 2-fold molar excess with the inhibitor has a s o 20 ,w of 203. Although this sedimentation coefficient does reflect compacting of the α 2 -macroglobulin structure compared to native α 2 -macroglobulin, it is not large enough to rule out significant protrusion of the proteases from within pockets in the α 2 -macroglobulin structure.

A nonantigenic covalent streptokinase-polyethylene glycol complex with plasminogen activator function.

A series of new, covalent polyethylene glycol (PEG)-streptokinase adducts were synthesized and characterized. PEGs with average molecular weights of 2,000, 4,000, and 5,000 were activated with carbonyldiimidazole and coupled to the protein under standardized reaction conditions. Steady-state kinetic analysis demonstrated comparable Km values for the activation of plasminogen by streptokinase, PEG-2-streptokinase, and PEG-4-streptokinase. The kcat values were somewhat decreased when PEG-2 or PEG-4 was coupled to the streptokinase. Activation by the PEG-5 adduct did not follow Michaelis-Menten kinetics under the conditions employed in this study. Plasmin activity obtained by incubating streptokinase derivatives with plasminogen also was studied as a function of time with each of the PEG-streptokinase derivatives. By this assay, incubations containing PEG-5-streptokinase and unmodified streptokinase demonstrated comparable activity while reaction mixtures containing PEG-2-streptokinase and PEG-4-streptokinase were slightly more active. Streptokinase incubated with plasminogen at a 1:1 molar ratio was extensively degraded after 30 min whereas PEG-2-streptokinase was resistant to plasmin cleavage. The derivatized proteins were radioiodinated and incubated in plastic microtiter plates that were coated with an immunoglobulin fraction containing antibodies to streptokinase. Binding of the PEG-streptokinase adducts was decreased by greater than 95% compared with unmodified streptokinase. Plasminogen activator complexes were formed by reacting the streptokinases with human plasminogen in vitro and the clearance studied in mice. Radioiodinated plasmin in complex with the PEG-streptokinase adducts cleared at a slower rate than did plasmin complexed with unmodified streptokinase. Catabolism of the protease still occurred by a mechanism that involved reaction with alpha 2-macroglobulin as has been described for nonderivatized streptokinase-plasminogen complex (Gonias, S. L., M. Einarsson, and S. V. Pizzo, 1982, J. Clin. Invest., 70:412-423). When more extensive derivatization procedures were utilized, PEG-2-streptokinase preparations were obtained that further prolonged the clearance of complexed 125I-plasmin; however, these adducts did not uniformly retain comparable activity. It is suggested that PEG-streptokinase complexes with greatly reduced antigenicity may be useful in the treatment of thrombotic disorders.

In vitro binding and in vivo clearance of human α2-macroglobulin after reaction with endoproteases from four different classes☆

The binding of human alpha 2-macroglobulin complexed with trypsin, papain, thermolysin and cathepsin-D to murine macrophages was studied at 4 degrees C. Similar dissociation constants (0.4 nM) were determined for all of the complexes except alpha 2-macroglobulin-cathepsin-D (0.7 nM). Radioiodinated alpha 2-macroglobulin-protease complexes were injected into mice, and the clearance studied. Native alpha 2-macroglobulin cleared slowly, as previously reported, while greater than 50% of the complexes formed with trypsin, papain and thermolysin cleared in less than 5 min. The clearance of alpha 2-macroglobulin-cathepsin-D was biphasic, suggesting that only about half the alpha 2-macroglobulin was present in a reacted complex.

Trypanosoma brucei gambiense: Growth in vitro of bloodstream forms inhibited by dichlorodiammineplatinum (II) compounds and hypolipidemic drugs

Infectious bloodstream forms of Trypanosoma brucei gambiense were grown in microcultures of murine bone marrow cells in 96-well tissue culture plates. Limiting dilution studies showed that fewer than 10 cultured trypanosomes developed into populations of about 5 X 10(4) parasites per well in a week. Bloodstream parasites were reisolated with high efficiency from mice infected with cultured parasites; fewer than 10 bloodstream parasites successfully established a trypanosome population in a microculture. Both the cis and trans isomers of dichlorodiammineplatinum (II) (cisplatin and transplatin) and a hypolipidemic agent, Wy 14643, were found to have activity against T. b. gambiense growing in microcultures. The minimum concentration of drug necessary to completely eliminate parasites from microcultures was 4 microM for cisplatin, 40 microM for transplatin, and 500 microM for Wy 14643. A preformed complex of cisplatin and bovine serum albumin and another hypolipidemic agent, chlofibric acid, were inactive. This culture system should be useful for rapid screening of large numbers of compounds for trypanocidal activity.