François Pochon

Three-dimensional reconstruction of a complex of human α2-macroglobulin with monomaleimido nanogold (Au1.4nm) embedded in ice

Cysteine 949 and glutamine 952 are known to be part of the thiol ester site of each of the four subunits of human alpha 2-macroglobulin (alpha 2M). The hydrolysis of this thiol ester bound to methylamine results in the incorporation of the amine and liberation of a free sulfhydryl group that can be specifically labeled. Therefore, a high-resolution marker specific for the sulfhydryl groups, the monomaleimido Nanogold (Au1.4nm) cluster was used to bind this amino acid. After cryoelectron microscopy, a three-dimensional reconstruction of the alpha 2M-Nanogold conjugates (alpha 2M-Au1.4nm) was achieved, revealing the internal location of the thiol ester sites in the transformed alpha 2M molecules. From this study we propose three possible locations for the cysteine 949.

Some consequences of the covalent and non-covalent binding modes of plasmin with α2-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.

The α2 macroglobulin/thrombin interaction in the presence of hydroxylamine

Abstract The influence of a primary amine, hydroxylamine, on the interaction between α 2 macroglobulin ( α 2 M) and thrombin was analyzed by electrophoretic and enzymatic methods. Hydroxylamine (final concentrations 0.01 M and 0.1 M) was added to the α 2 M solution 3 to 5 min before thrombin. In these conditions hydroxylamine had no direct influence on α 2 M itself. The inhibition of thrombin activity by α 2 M was still possible and α 2 M/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 α 2 M polypeptide chains cleaved by thrombin was the same (50%). In conclusion, hydroxylamine does not prevent the formation of α 2 M/thrombin complexes, but it reduces the covalent binding of the enzyme to the inhibitor in a concentration dependant fashion, leading to the formation of “abnormal” complexes.

Functional α2-macroglobulin half-molecules induced by cadmium

Abstract Human α 2 -macroglobulin can be reversibly dissociated by Cd 2+ at low ionic strength in half-molecules which retain their ability to bind tightly plasmin and chymotrypsin. The steady state kinetic parameters of these proteinases towards chromogenic substrates when bound to half-molecules are not greatly different from those determined for these enzymes linked to whole α 2 M molecules. Cd 2+ can also induce the dissociation of plasmin- and chymotrypsin - α 2 M complexes into proteinase-α 2 M half-molecule conjugates. These results, taken with the fact that monomeric units of α 2 M cannot bind these proteinases, strongly suggest that each active site of α 2 M consists in a specific arrangement of two monomeric units linked by disulfide bridges.

Structural arrangement in the α2-macroglobulin—thrombin complex

The cysteine sulfhydryl groups of α2‐macroglobulin (α2M) 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 α2M to fluorescein isothiocyanate‐labeled thrombin. The thrombin‐α2M 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 α2M binding sites of thrombin are quite similar, whatever covalent or non‐covalent binding occurs.

Dissociation of α2 M-macroglobulin into functional half-molecules by mild acid treatment

Abstract A slight decrease in pH below neutrality causes the dissociation of α2-macroglobulin(α2M) 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 α2M 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 α2M 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 α2M complexes. These results are consistent with the idea that α2M-half molecules on chymotrypsin binding undergo a conformational change. This change can be observed by electron microscopy.

Covalent and non-covalent interaction of chymotrypsin with α2-macroglobulin

The pattern of covalent crosslinking between human α2‐macroglobulin (α2M) and chymotrypsin has been investigated by chromatography and polyacrylamide gel electrophoresis in denaturing medium. Reaction with a single mol of chymotrypsin per mol α2M results in the formation of a 95% covalent 1:1 chymotrypsin‐α2M complex and in the proteolytic cleavage of both 180 kDa monomers in one α2M subunit. Proteolytic cleavage in the other α2M subunit requires the presence of a second mol of chymotrypsin; part (20%) of the protease in the 2:1 chymotrypsin‐α2M complex thus formed appears to be non‐covalently bound to the α2M chains. Covalent binding is abolished when the reaction of α2M 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 α2M.

Mechanisms of Facilitated CO 2 and H + Diffusion in Protein Solutions

Facilitated diffusion of CO2 under steady-state conditions is based on two simultaneous processes (a) diffusion of HCO 3 − and (b) an equivalent H+ flux that is brought about by a facilitated H+ diffusion. We have previously shown that, in this latter process, proteins and other buffers can function as proton carriers in such a powerful way that facilitated H+ fluxes at neutral pH values are 1000–10,000 times greater than the fluxes of free protons (Gros and Moll, 1974; Gros et al., 1976). Inside cells, where soluble proteins and other mobile buffers are present, proton fluxes should almost exclusively occur by such a facilitated transport process. Our main aim in this study was to understand the mechanisms underlying these facilitated proton fluxes, and we have used the measurement of facilitated CO2 diffusion as a tool to obtain quantitative information on proton transport in protein solutions.

Localization of the proteinase-induced thiol groups in α2-macroglobulin

Abstract Free thiol groups released on proteolytic attack of α 2 -macroglobulin by trypsin or chymotrypsin bind covalently to thiopropyl-Sepharose, indicating that they are located at the surface of the complexes. These cysteine sulfhydryl groups appear to be in contact with the α 2 M-bound proteases from singlet-singlet energy transfer measurements between fluorescein isothiocyanate-labeled proteinases and N-(iodoacetylaminoethyl)-5-naphtylamine-1-sulfonic acid-labeled thiols in α 2 -macroglobulin.

Effect of carcinogens on DNA: Action of 7-bromomethylbenz (a) anthracene

Summary Factors governing the substitution of DNA by the carcinogen 7-bromomethylbenz(a)anthracene are discussed and the mechanism of reaction elaborated. Different kinds of double helix show quite different reactivity and reaction appears to be rather specific for the B form. Cross linking can also occur between the two strands, from a substituted guanine (or adenine) residue in one strand to a deoxypyrimidine nucleoside in the other. The cross-linked product has been isolated and certain characteristics described.