Covalent thrombin-.alpha.2-macroglobulin complexes. Evidence for bivalent crosslinking of inhibitor chains by a single enzyme molecule

Wang, Dalton; Yuan, Anna I.; Feinman, Richard D.

doi:10.1021/bi00307a042

Cited by 33 publications

(22 citation statements)

References 17 publications

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“…Strikingly, the fibrinolytic activity is decreased during this period. In the present work, we demonstrate by PAGE analysis that PZP interacts with t-PA within 1 h of incubation at room temperature, whereas a 2 -M reacts over a longer period of up to 18 h. Formation of a -macroglobulint-PA complexes was evidenced by a number of observations, as follows: (a) PZP and <z 2 -M showed changes in their mobility rates in non-denaturing PAGE comparable to those observed with chymotrypsin (2,3,28); (b) both PZP and a 2 -M, formed complexes of molecular size >360 kDa in agreement with their covalent binding to t-PA, as reported for other proteinases (30)(31)(32); and (c) PZP underwent a specific cleavage of the bait region with rapid appearance of fragments of 85-90 kDa as judged by reducing SDS-PAGE. In contrast, this proteolytic attack on a 2 -M as found to be slow, requiring several hours of incubation with t-PA for generation of an appreciable amount of fragments of 85-90 kDa (2,28).…”

Section: Discussionsupporting

confidence: 71%

Interaction of Human Tissue Plasminogen Activator (t-PA) with Pregnancy Zone Protein: A Comparative Study with t-PA- 2 -Macroglobulin Interaction

Sánchez¹,

Chiabrando

Guglielmone

et al. 1998

Journal of Biochemistry

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Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to alpha2-macroglobulin (alpha2-M). Interactions of tissue plasminogen activator (t-PA) with PZP and alpha2-M were both investigated in vitro and the complexes were analyzed by polyacrylamide gel electrophoresis (PAGE). The results demonstrated that PZP-t-PA complex formation was evident within 1 h of incubation, whereas alpha2-M-t-PA complexes were formed after 18 h. Conclusions were supported by the following evidence: (i) PZP and alpha2-M complexes revealed changes of the mobility rate in non-denaturing PAGE, similar to those observed with alpha-Ms-chymotrypsin; (ii) both PZP and alpha2-M formed complexes of molecular size >360 kDa by SDS-PAGE, in accordance with the covalent binding of t-PA, which was previously reported for other proteinases; and (iii) PZP underwent a specific cleavage of the bait region with appearence of fragments of 85-90 kDa as judged by reducing SDS-PAGE. In contrast, the proteolytic attack on alpha2-M was found to occur more slowly, requiring several hours of incubation with t-PA for generation of an appreciable amount of fragments of 85-90 kDa. The appearance of free SH-groups of alpha-Ms was further investigated by titration with 5, 5'-dithiobis(2-nitrobenzoic acid). The maximal level of SH-groups raised was 3.9 mol/mol of PZP and 3.5 mol/mol of alpha2-M, indicating approximately one SH-group for each 180-kDa subunit. Finally, t-PA activity in PZP-t-PA complex was evaluated by measuring the hydrolysis of the chromogenic substrate Flavigen t-PA. Our results revealed that prolongation of the incubation period of this complex increased t-PA-mediated hydrolysis of Flavigen t-PA until a plateau was reached, approximately between 60 and 120 min. The present study suggests that PZP, by binding to t-PA, may contribute to the control of the activity of proteinases derived from fibrinolytic systems.

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Section: Discussionsupporting

confidence: 71%

Interaction of Human Tissue Plasminogen Activator (t-PA) with Pregnancy Zone Protein: A Comparative Study with t-PA- 2 -Macroglobulin Interaction

Sánchez¹,

Chiabrando

Guglielmone

et al. 1998

Journal of Biochemistry

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“…This idea is also supported by experiments performed by Gonias & Pizzo (1983a, b), which showed high-Mr enzyme-containing species in the reaction of trypsin with native a2M but not with dissociated half-molecules. The fact that the formation of these complexes is decreased in reactions with enzymes in which the lysine-residue amino groups have been blocked (Wang et al, 1984) suggests that the linkages are an amide of the type involved in univalent complexes (Sottrup-Jensen et al, 1983).…”

Section: Discussion Multivalent Enzyme-a2m Complexesmentioning

confidence: 99%

“…To simplify the interpretation of the kinetic data, the behaviour of the thrombin-cc2M system on SDS/polyacrylamide-gel electrophoresis (Wang et al, 1983(Wang et al, , 1984 is briefly summarized. There are five major bands.…”

Section: Sds/polyacrylamide-gel Electrophoresis Of Thrombin-a2m Complmentioning

confidence: 99%

“…Band 2 is usually attributed to the half-molecule with covalently bound enzyme. Band 3 is assumed to be a covalently cross-linked a2M species of the size of the entire a2M molecule (Mr greater than 750000) although it has anomalously high mobility (Wang et al, 1984). Bands 4 and 5 have not been characterized but are believed to be separated by similar large differences in mass.…”

Section: Sds/polyacrylamide-gel Electrophoresis Of Thrombin-a2m Complmentioning

confidence: 99%

“…The potential for covalent-bond formation appears to be great, and evidence has been presented (Wang et al, 1983(Wang et al, , 1984 that bound enzymes are capable of forming more than one covalent bond to the subunits of a2M. This is the likely explanation for enzyme-ox2M complexes of very slow mobility seen on dissociating gels, which, in some cases, account for the majority of the covalent complexes (Wang et al, 1983;van der Graaf et al, 1984).…”

Section: Introductionmentioning

confidence: 99%

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Kinetics of the reaction of thrombin and α2-macroglobulin

Feinman¹,

Yuan²,

Windwer³

et al. 1985

Biochemical Journal

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The kinetics of the reaction of alpha 2-macroglobulin (alpha 2M) with human thrombin were studied by recording the appearance of thiol groups spectrophotometrically and by measuring the distribution of protein species by denaturing non-reducing gel electrophoresis. The goals were to study the relation between the formation of various covalent enzyme-inhibitor complex species and the appearance of free thiol, and from the kinetic analysis, to try to characterize the chemical nature of the protein complexes. The kinetics of thiol-group release were observed to be biphasic, the early phase showing second-order behaviour, results consistent with previous reports in the literature. The observed second-order rate constant for thiol-group release was found to be faster than the second-order rate constant for the disappearance of the band corresponding to native alpha 2M on gel electrophoresis. This may be a reflection of the multiple products formed from the thioester. Alternatively, it is possible that covalent-bond formation is slower than some enzyme-induced change in the thioester centre, and this may be suggestive evidence for a reactive alpha 2M centre that does not contain an intact thioester. The kinetics of covalent-bond formation were found to be consistent with the internal cross-link of several alpha 2M chains by the bound proteinase, providing further evidence that the very-high-Mr species seen on gels may arise from dimers of the alpha 2M molecule held together by covalent bonds to the enzyme.

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