Some consequences of the covalent and non-covalent binding modes of plasmin with alpha 2-macroglobulin.

Analysis of plasmin-alpha 2-macroglobulin interactions by polyacrylamide gel electrophoresis showed that both the light and heavy chains of the proteinase have covalent links with the inhibitor. This covalent binding occurs with a 95 +/- 5% yield and can be abolished in the presence of hydroxylamine without modification of the plasmin-alpha 2-macroglobulin stoichiometry, the extent of the 180-kDa peptide chain cleavage and the generation of the -SH groups. However, these two different binding modes greatly influence the enzymatic properties of the proteinase as well as the occupancy by an other proteinase molecule of the free binding site of the (1:1) plasmin-alpha 2-macroglobulin complex. Non-covalently bound plasmin is more active on synthetic substrates and interacts more tightly with the basic pancreatic trypsin inhibitor than the covalently bound enzyme. Furthermore, the former complex incorporates significantly more chymotrypsin than the latter. The incorporation of chymotrypsin influences the catalytic properties of plasmin within the ternary complex.

Dissociation of alpha 2 M-macroglobulin into functional half-molecules by mild acid treatment.

A slight decrease in pH below neutrality causes the dissociation of alpha 2-macroglobulin (alpha 2M) into dimers formed of two disulfide-bonded subunits. Half-dissociation occurs at pH 6.30 (50 mM NaCl), as determined by gel filtration analysis. The dissociation can be reversed either by increasing the pH or the ionic strength. The ability of alpha 2 M half-molecules at pH 5.75 to bind chymotrypsin is not too different from that of the whole molecule at pH 7.5. Furthermore, the steady-state kinetic parameters toward chromogenic substrate of chymotrypsin bound to alpha 2 M half and whole molecules are quite identical. Likewise, the accessibility of trypsin toward soybean trypsin inhibitor is also fairly similar when involved in half or whole alpha 2 M complexes. These results are consistent with the idea that alpha 2 M-half molecules on chymotrypsin binding undergo a conformational change. This change can be observed by electron microscopy.

The two alpha 2-macroglobulin-bound trypsin molecules have different affinities for the basic pancreatic trypsin inhibitor.

We have investigated the enzymatic properties of alpha 2-macroglobulin-bound porcine trypsin using a substrate: Z-Gly-Gly-Arg-p-nitroanilide and two inhibitors: p-aminobenzamidine and basic pancreatic trypsin inhibitor. The ternary alpha 2-macroglobulin-(trypsin)2 complex behaves like a mixture of two enzymes which bind basic pancreatic trypsin inhibitor with widely different affinities (Ki = 0.11 microM and 23 microM). About one-half of the trypsin molecules of the ternary complex are covalently bound to alpha 2-macroglobulin. Preparation of the complex in the presence of hydroxylamine prevents covalent bond formation, but the two trypsins of this artificial complex still exhibit large differences in affinity for basic pancreatic trypsin inhibitor. The trypsin molecules of the ternary complex also exhibit small differences in their affinity for Z-Gly-Gly-Arg-p-nitroanilide and p-aminobenzamidine.

Three-dimensional architecture of human alpha 2-macroglobulin transformed with methylamine.

A frozen-hydrated sample embedded in vitreous ice of human alpha 2-macroglobulin transformed by methylamine was imaged by cryoelectron microscopy and reconstructed in three dimensions. In the reconstruction, the cage-like architecture of this protease inhibitor is fully revealed with a clear visualization of two lozenge-shaped lateral walls connected by thin bridges. The shape and dimensions of the internal cavity normally containing the trapped protease(s) is described. The possible locations of the thiol ester sites and inter-subunit connections are also discussed.

Ultrastructural and anti-proteinase activity modifications of human alpha 2-macroglobulin induced by zinc and other divalent cations.

Native tetrameric alpha 2-macroglobulin molecules (alpha 2M) can be converted into a population of dimers by incubation with various divalent cations such as Zn, Cd, Mg, Cu, Ni, Co. This dissociation is completed within 30 min at 37 degrees C. These dimers have a characteristic shape and a size of about 16 X 8 nm, and appear to be the half of the native alpha 2M molecule which has a clear tetrameric structure as seen in the electron microscope. At room temperature or below, dimers obtained with 5 to 100 mM Zn++ can reassociate in long linear polymers which display a regular chain-like arrangement and a helical periodicity. The structural characteristics of this polymer are described. The trypsin inhibitory capacity of Zn++-treated alpha 2M has been studied in an attempt to correlate its Zn++-induced conformational changes with its functional modifications.

Interaction of alpha 2-macroglobulin-bound thrombin with hirudin.

The human thrombin bound to alpha 2-macroglobulin (alpha 2 M) in a 1:1 stoichiometry is still able to interact with one of its specific inhibitors, hirudin. The dissociation constant of the complex hirudin--alpha 2M-bound thrombin is 1 X 10(-7) M, whatever the mode of thrombin binding, covalent or non-covalent.

Structural arrangement in the alpha 2-macroglobulin--thrombin complex.

The cysteine sulfhydryl groups of alpha 2-macroglobulin (alpha 2M) generated upon thrombin complex formation are in contact with the proteinase surface as evidenced by singlet--singlet energy transfer measurements from N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid-labeled thiol functions of alpha 2M to fluorescein isothiocyanate-labeled thrombin. The thrombin-alpha 2M binding is normally covalent, but the presence of hydroxylamine during the reaction leads to the formation of a non-covalent complex. The transfer energy determinations show that the alpha 2M binding sites of thrombin are quite similar, whatever covalent or non-covalent binding occurs.

Covalent and non-covalent interaction of chymotrypsin with alpha 2-macroglobulin.

The pattern of covalent crosslinking between human alpha 2-macroglobulin (alpha 2M) and chymotrypsin has been investigated by chromatography and polyacrylamide gel electrophoresis in denaturing medium. Reaction with a single mol of chymotrypsin per mol alpha 2M results in the formation of a 95% covalent 1:1 chymotrypsin-alpha 2M complex and in the proteolytic cleavage of both 180 kDa monomers in one alpha 2M subunit. Proteolytic cleavage in the other alpha 2M subunit requires the presence of a second mol of chymotrypsin; part (20%) of the protease in the 2:1 chymotrypsin-alpha 2M complex thus formed appears to be non-covalently bound to the alpha 2M chains. Covalent binding is abolished when the reaction of alpha 2M with the protease is carried out in the presence of hydroxylamine. A single mol of the protease is then able to cleave all four 180 kDa monomers in alpha 2M.

Specific fluorescent labeling of alpha 2-macroglobulin-bound proteases: accessibility and localization of their active site within the complex.

Pyrenebutylmethylphosphonofluoridate reacts with trypsin and elastase to yield a conjugate with a stoichiometry of one fluorescent label per enzyme molecule as already observed with chymotrypsin. The kinetics of inactivation indicate that the serine active center of the proteases is involved in the labeling reaction. The binding of the proteases to alpha 2-macroglobulin does not modify the specificity of the reaction but drastically diminishes the labeling rate which also depends upon alpha 2-macroglobulin protease binding ratio. Dynamic quenching of the conjugated pyrene moiety by acrylamide, and iodide ions is markedly reduced upon reaction of the protease with alpha 2-macroglobulin, indicating a reduced accessibility of the protease active center in the complex. Singlet--singlet energy transfer measurements from the donor pyrene labeled active center of the proteases to the alpha 2-macroglobulin acceptor labeled thiol groups which are liberated upon protease fixation, gave a rough estimate of the distance (about 25 A) between the active center of the two alpha 2-macroglobulin bound protease molecules.

Rotational diffusion of Escherichia coli ribosomes. I. - Free 70 S, 50 S and 30 S particles.

The rotational brownian diffusion of Escherichia coli ribosomes has been studied by following the transient dichroism generated by optical excitation of a covalent probe into its triplet state. The induced absorption anisotropy decays exponentially with characteristic correlation times: 2.5 microseconds, 1.6 microseconds and 1.1 microseconds for the 70S ribosome and the 50S and 30S subparticles respectively. The corresponding Stokes radii are in the same order, 133 A, 115 A and 103 A. The hydrodynamic properties are discussed in terms of an ellipsoidal shape of the ribosome particles.

Rotational diffusion of Escherichia coli ribosomes. II. - Ribosome attachment to polyurydilic acid.

Ribosome attachment to poly(U) has been studied by following the rotational diffusion of polyribosomes in solution. On the average, 13-17 and 50 nucleotides are found to be associated with 30S and 70S ribosome respectively. For an equal length of poly(U), the number of particles in a 30S polysome is four times that in a 70S polysome. The results are consistent with a structure of the polysome in which individual ribosomes are in close contact.

Conformational change at the active center of serine-proteases upon their binding to alpha 2-macroglobulin.

The inhibition rates and spectral characteristics of 2 probes specific for the active-site serine residue of proteases were examined for evidence of conformational change of the proteases upon their binding to alpha 2-macroglobulin (alpha 2M). Elastase, chymotrypsin, trypsin, and plasmin were reacted with (7-nitrobenz-2-oxa-1,3-diazole) aminoethyl- and aminopentyl methylphosphonofluoridate. The inhibition rate constants depend on the chain length of the aminoalkyl moiety of the probe and range from 10(5) to 10(4) M-1 min-1 for elastase and chymotrypsin. They are significantly modified when the proteases are stoichiometrically bound to alpha 2M. The absorption maximum of the chromophore appears in the range of 460-470 nm and 475-480 nm for the aminoethyl- and aminopentyl- conjugates, respectively. The fluorescence emission is maximal around 530 nm with a low quantum yield of about 3%. These spectral characteristics are altered in different ways by the covalent or non-covalent binding mode of the protease to alpha 2M. Finally, the CD spectrum of the NBD aminoethyl and aminopentyl elastase and chymotrypsin conjugates exhibits intense optical activity in the absorbing band of the NBD-moiety. These chiral properties are greatly altered upon binding of the protease to alpha 2M. All these results strongly suggest a conformational change in the protease at its active center upon its binding to alpha 2M; this conformational change could be taken into account to explain the alteration of the catalytic properties of the alpha 2M-bound proteases.

Structure-activity relationships in a series of newly synthesized 1-amino-substituted ellipticine derivatives.

The synthesis of a series of 1-amino-substituted pyrido[4,3-b]carbazole derivatives, based on the substitution of corresponding 1-chloroellipticines, is reported. The cytotoxic properties on tumor cells grown in vitro, the in vivo acute toxicity of the most potent in vitro cytotoxic compounds, and the antitumor properties toward the L1210 leukemia system are described. No correlation between the apparent association constant to DNA and the in vitro cytotoxicity or the in vito antitumor efficiency could be observed in this series. 9-Hydroxylated derivatives were more cytotoxic in vitro than the corresponding 9-methoxylated compounds. However, their antitumor efficiencies on the in vivo experimental systems do not confirm the advantage of demethylation. The presence of a [(dialkylamino)alkyl]amino side chain at the 1 position of ellipticines increases the antitumor potency: 1-[[3-(diethylamino)propyl]amino]-5,11-dimethyl-6H-pyrido[4,3-b]carbazole (5) is a very potent antitumor compound (% ILS of 134 on the L1210 leukemia system).

Structural arrangement of the proteinase binding sites in human alpha 2-macroglobulin.

Using singlet-singlet energy transfer measurements with labeled-chymotrypsin-alpha 2-macroglobulin complexes, we find that the two proteinase binding sites of alpha 2-macroglobulin are separated from each other by 44 A. The free thiol groups generated upon reaction of alpha 2-macroglobulin with trypsin or chymotrypsin react with thiopropyl Sepharose, indicating that they are located at the surface of the complexes. Singlet-singlet energy transfer experiments from labeled proteinases to labeled thiols of alpha 2-macroglobulin show that the thiol groups are in close contact with the proteinase molecules whether the latter are covalently or noncovalently bound to alpha 2-macroglobulin. In addition, they are remote from the association interface between the Mr = 360,000 halves of alpha 2-macroglobulin. Using the same approach we demonstrate that the active sites of chymotrypsin molecules are separated by a distance of at least 20 A from the thiols group of each alpha 2-macroglobulin subunit.

Heparin and the progressive anti-thrombin activity of alpha 2-macroglobulin.

Interactions between alpha 2-macroglobulin (alpha 2M) and thrombin have been studied by spectroscopic, isotopic and electrophoretic methods in presence or in absence of heparin. It is shown that thrombin binds to alpha 2M in a 1:1 ratio. Fluorescamin labelled heparin of Mr 7 000 interacts with thrombin to form a 2:1 molar complex. This complex does not bind to alpha 2M and is unable to achieve any proteolytic cleavage of this protein. In contrast the interaction of alpha 2M with chymotrypsin is not significantly affected by the mucopolysaccharide. Moreover, heparin is unable to react with alpha 2M bound thrombin.

The alpha 2 macroglobulin/thrombin interaction in the presence of hydroxylamine.

The influence of a primary amine, hydroxylamine, on the interaction between alpha 2 macroglobulin (alpha 2M) and thrombin was analyzed by electrophoretic and enzymatic methods. Hydroxylamine (final concentrations 0.01 M and 0.1 M) was added to the alpha 2M solution 3 to 5 min before thrombin. In these conditions hydroxylamine had no direct influence on alpha 2M itself. The inhibition of thrombin activity by alpha 2M was still possible and alpha 2M/thrombin complexes were observed. However the rate of inhibition of the clotting activity of thrombin was diminished in function of the hydroxylamine concentration. The complexes obtained in the absence of the nucleophylic agent were resistant to SDS dissociation, whereas those obtained in the presence of hydroxylamine were dissociated by SDS. In both cases, the amount of alpha 2M polypeptide chains cleaved by thrombin was the same (50%). In conclusion, hydroxylamine does not prevent the formation of alpha 2M/thrombin complexes, but it reduces the covalent binding of the enzyme to the inhibitor in a concentration dependent fashion, leading to the formation of "abnormal" complexes.

Intercalative binding to DNA of new antitumoral agents: dipyrido [4,3-b] [3,4-f] indoles.

The study of new intercalating DNA agents has led to the synthesis of dipyrido [4,3-b] [3,4-f] indole derivatives. These compounds bind preferentially to helical DNA and their affinity constants range from 10(6) to 10(7) M-1. The most active derivative against L1210 mouse leukemia produces viscosity changes in sheared fragments of linear DNA and in circular PM2 DNA on binding, indicating an intercalation process. The unwinding angle is estimated to be 18(0). This active derivative binds preferentially to A-T base pairs.