The interaction of a naphthalene dye with , free or bound to trypsin

Jacquot-Armand, Y; Krebs, Guillaume

doi:10.1016/0005-2795(73)90154-2

Cited by 17 publications

(5 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…TNS Fluorescence Studies. Previous studies have reported that the fluorescence of ANS is enhanced in the presence of porcine a2M or the trypsin-a2M complex (Jacquot-Armand & Krebs, 1973). In the present study, the interactions of several fluorescent probes with a2M, methylamine-treated a2M, and a2M-protease complexes were investigated.…”

Section: Resultsmentioning

confidence: 97%

“…At this time, the interaction of TNS with a2M has not been fully characterized. Previous studies on the interaction of ANS with a2M and the trypsin-a2M complex indicated a slight increase in the quantum yield of the dye when bound to the complex vs. that bound to free a2M (Jacquot-Armand & Krebs, 1973). Although the exact nature of the conformational change occurring in methylamine-treated a2M and in the a2M-protease complex that causes an increased TNS fluorescence is not known, it does appear that the changes in TNS fluorescence are reflecting alterations in a2M conformations.…”

Section: Discussionmentioning

confidence: 97%

See 1 more Smart Citation

Kinetics of the conformational alterations associated with nucleophilic modification of .alpha.2-macrobglobulin

Strickland¹,

Bhattacharya²,

Olson³

1984

Biochemistry

View full text Add to dashboard Cite

The effect of nucleophilic modification of alpha 2-macroglobulin (alpha 2M) with methylamine on the kinetics of sulfhydryl exposure was investigated. The generated sulfhydryl groups were detected with 4,4'-dithiodipyridine. The bimolecular rate constant for sulfhydryl exposure was determined to be 11.6 +/- 0.8 M-1 s-1 at 30 degrees C and pH 8.0. Treatment of alpha 2-macroglobulin with methylamine or proteases, such as plasmin and trypsin, results in a substantial increase in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonic acid. This probe was used to monitor the kinetics of the conformational change occurring in alpha 2-macroglobulin upon treatment with methylamine. It was found that the conformational change did not occur simultaneously with the cleavage of the thiol ester bonds by the nucleophile but, rather, the conformational alterations occurred following a lag phase. The data are consistent with a mechanism requiring the random cleavage of two thiol ester bonds of a dimeric unit in the molecule prior to the unimolecular process representing the conformational change. According to this model, the two dimeric units present in alpha 2M act as independent entities. On the basis of the best fit with the model, the bimolecular rate constant, for hydrolysis of the thiol ester bonds, was determined to be 11.9 +/- 0.7 M-1 s-1, while the rate constant for the conformational change was (9.7 +/- 2.0) X 10(-3) s-1. The measured rate of conformational change is rate limited by thiol ester cleavage at lower methylamine concentrations. The conformational change, measured with this fluorescence probe, approximately parallels the loss of trypsin binding activity of alpha 2-macroglobulin, measured by resistance of the bound trypsin to soybean trypsin inhibitor. A much slower loss of plasmin binding activity was observed than was found for trypsin, suggesting that the structural requirements on alpha 2M for the interaction with plasmin are disrupted much more slowly than the structural requirements for trypsin binding upon methylamine treatment of the molecule.

show abstract

Section: Resultsmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 97%

Kinetics of the conformational alterations associated with nucleophilic modification of .alpha.2-macrobglobulin

Strickland¹,

Bhattacharya²,

Olson³

1984

Biochemistry

View full text Add to dashboard Cite

show abstract

“…This site has been tentatively identified as a thiol ester cross-link between cysteine and glutamic acid residues that are separated by two residues in the polypeptide chain (Sottrup-Jensen et al, 1981a;Howard, 1981). Cleavage of the thiol ester bonds results in inactivation of a2M, exposure of four thiol groups per molecule, and a significant conformational change in the a2M molecule (Barrett et al, 1979;Marelis et al, 1969;Jacquot-Armand & Krebs, 1973). This latter observation prompted us to determine whether a change in protein intrinsic fluorescence might occur and correlate with the reactions that change the conformation of a2M.…”

mentioning

confidence: 99%

Effect of protease binding by .alpha.2-macroglobulin on intrinsic fluorescence

Straight¹,

McKee²

1982

Biochemistry

View full text Add to dashboard Cite

We have evaluated intrinsic protein fluorescence as a method for investigating the reactions of alpha 2-macroglobulin (alpha 2M) with proteases and amines. Changes in fluorescence intensity of alpha 2M in the presence of proteases and amines were shown to correlate with structural and functional changes in the alpha 2M molecule. By intrinsic fluorescence we found that 2 mol of trypsin bound to 1 mol of alpha 2M whereas thrombin and plasmin each bound in a stoichiometry closer to 1:1. Studies showed that changes in fluorescence caused by ammonium ion paralleled the loss of the ability of alpha 2M to protect trypsin from soybean trypsin inhibitor. The exposure of sulfhydryl groups on alpha 2M by a small organic amine (methylamine) also correlated with fluorescence change that could be quantitatively eliminated by prior reaction of alpha 2M with trypsin. Cleavage of alpha 2M by four serine proteases (plasmin, thrombin, trypsin, and elastase) as determined by sodium dodecyl sulfate gel electrophoretic analyses and the binding of plasmin and thrombin as measured by macromolecular inhibitor assays corresponded to the increase in fluorescence intensity. In addition, the rate of thrombin inhibition for clotting fibrinogen was the same as the rate of fluorescence change observed when thrombin was incubated with alpha 2M. Our results indicate that intrinsic protein fluorescence is an easy and rapid technique for assessing both qualitative and quantitative aspects of protease-alpha 2M interactions.

show abstract

“…The quaternary structure consists of two noncovalently bound protomers; each protomer is made up of two subunits covalently linked by two disulfide bonds (4). When exposed to an endoprotease, a limited proteolysis of a2M occurs at a site called the "bait" region, located near the middle of the protein, resulting in a change in its structure (5)(6)(7). The structural transformation has been identified by increases in the sedimentation coefficient (8) and in the electrophoretic mobility (9) and by decreases in the radius of gyration (10) and in the Stokes' radius (11).…”

mentioning

confidence: 99%

Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

Bretaudiere

Tapon‐Bretaudière

Stoops

1988

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Well-preserved structures of native and achymotrypsin-bound a2-macroglobulin were obtained by electron microscopy. Computer processing of these images has shown that the native structure has the shape of a padlock 19 nm long. It is proposed that the native a2-macroglobulin consists of the juxtaposition of two protomers with one protomer shaped like a distorted letter "S" and with the other its reverse image, to form a binding site between the two protomers near the bottom of the complex. On cleavage of the subunits with chymotrypsin, the native structure condenses to 16.7 nm and rearranges so that the interaction between the protomers is near the middle. Two images of the a2-macroglobulin-chymotrypsin conijugate were obtained. We suggest that these images represent the end and side view of this complex. Based on the manner in which the native structure is assembled, we propose that the proteolyzed form of a2-macroglobulin is functionally asymmetric in that both protease binding sites reside on the same half of the complex. a2-Macroglobulin (a2M) is one of the major antiproteases found in the plasma of vertebrates. It has the capacity to inhibit not only endoproteases normally present in the plasma but also proteases from other origins (1). a2M, isolated from human plasma, is a large glycoprotein (Mr, 725,000) composed of four identical subunits (Mr, 180,000) (2, 3). The quaternary structure consists of two noncovalently bound protomers; each protomer is made up of two subunits covalently linked by two disulfide bonds (4). When exposed to an endoprotease, a limited proteolysis of a2M occurs at a site called the "bait" region, located near the middle of the protein, resulting in a change in its structure (5-7). The structural transformation has been identified by increases in the sedimentation coefficient (8) and in the electrophoretic mobility (9) and by decreases in the radius of gyration (10) and in the Stokes' radius (11). The inactivated protease may form a covalently bound complex with a2M by reacting with an intramolecular thiol ester linkage between glutamine and cysteine residues. Even though there are four "bait" sites available, it has been proposed that a2M has two independent and identical proteinase binding sites (12)(13)(14). By using energy transfer experiments, Pochon et al. (12) demonstrated that the two sites are 4.4 nm apart. The binding of 2 mol of proteases per mol of a2M has been observed only with smaller proteases like trypsin and chymotrypsin (Mr, 25,000); larger proteases like plasmin (Mr, 81,000) display a 1:1 molar binding ratio (15).There have been conflicting reports relating the various structures of a2M obtained by electron microscopy to the native and protease bound forms. This discrepancy seems to have resulted from the study of a2M that had undergone proteolysis during its isolation (16). Early studies reported the presence of two forms (7,(17)(18)(19): an ovate structure shaped like two crescents facing each other with a bar in the center (the "closed" form) and a ...

show abstract

The interaction of a naphthalene dye with , free or bound to trypsin

Cited by 17 publications

References 21 publications

Kinetics of the conformational alterations associated with nucleophilic modification of .alpha.2-macrobglobulin

Kinetics of the conformational alterations associated with nucleophilic modification of .alpha.2-macrobglobulin

Effect of protease binding by .alpha.2-macroglobulin on intrinsic fluorescence

Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

Contact Info

Product

Resources

About