Binding of anhydrotrypsin to α2-macroglobulin

Sayers, C A; Barrett, Alan J.

doi:10.1042/bj1890255

Cited by 28 publications

(8 citation statements)

References 12 publications

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“…Human TSG-6 was expressed and purified, also as described before (23,29). Anhydrotrypsin-Sepharose was prepared essentially as described previously (30,31). Protein concentrations were determined spectrophotometrically at 280 nm using theoretical extinction coefficients (GPMAW software, Lighthouse Data).…”

Section: Methodsmentioning

confidence: 99%

The TSG-6/HC2-mediated Transfer Is a Dynamic Process Shuffling Heavy Chains between Glycosaminoglycans

Sanggaard

Scavenius

Rasmussen

et al. 2010

Journal of Biological Chemistry

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The heavy chain (HC) subunits of the bikunin proteins are covalently attached to a single chondroitin sulfate (CS) chain originating from bikunin and can be transferred to different hyaluronan (HA) molecules by TSG-6/HC2. In the present study, we demonstrate that HCs transferred to HA may function as HC donors in subsequent transfer reactions, and we show that the CS of bikunin may serve as an HC acceptor, analogous to HA. Our data suggest that TSG-6/HC2 link HCs randomly on the CS chain of bikunin, in contrast to the ordered attachment observed during the biosynthesis. Moreover, the results show that the transfer activity is indifferent to the new HC position, and the relocated HCs are thus prone to further TSG-6/HC2-induced transfer reactions. The data suggest that HCs may be transferred directly from HA to HA without the involvement of the bikunin CS chain. The results demonstrate reversibility of the interactions between HCs and glycosaminoglycans and suggest that a dynamic shuffling of the HCs occur in vivo.

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Section: Methodsmentioning

confidence: 99%

The TSG-6/HC2-mediated Transfer Is a Dynamic Process Shuffling Heavy Chains between Glycosaminoglycans

Sanggaard

Scavenius

Rasmussen

et al. 2010

Journal of Biological Chemistry

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“…Plasminogen (Deutsch and Mertz, 1970) and ␣ 2 M (Salvesen and Enghild, 1993) were purified as described previously. Anhydrotrypsin was prepared as described by Sayers and Barrett (1980).…”

Section: Methodsmentioning

confidence: 99%

“…Pro-plasma CPB was unable to compete for binding to ␣ 2 M even at a 1000-fold molar excess confirming that the pro-plasma CPB component does not bind to ␣ 2 M. Similarly, no competition was observed using anhydrotrypsin as the competing ligand. Since it is known that anhydrotrypsin binds to ␣ 2 M (Sayers and Barrett, et al, 1980), the data suggest that plasma CPB and anhydrotrypsin bind to different sites on the ␣ 2 M molecule.…”

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confidence: 96%

Activated Human Plasma Carboxypeptidase B Is Retained in the Blood by Binding to α2-Macroglobulin and Pregnancy Zone Protein

Valnickova

Thøgersen

Christensen

et al. 1996

Journal of Biological Chemistry

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A 66-kDa glycosylated carboxypeptidase, plasma procarboxypeptidase B (pro-plasma CPB), has recently been identified in human blood (Eaton, D. L., Malloy, B. E., Tsai, S. P., Henzel, W., and Drayna, D. (1991) J. Biol. Chem. 266, 21833-21838). The pro-enzyme binds to plasminogen and the active enzyme is specific for COOHterminal Lys or Arg residues. These properties implicate a role in the fibrinolytic or coagulation system. However, we show that the molecular mass of the active plasma CPB is approximately 36 kDa, which is below the glomerular filtration limit. Since activated plasma CPB no longer binds plasminogen, the active enzyme may not be retained in the circulation. To investigate this, we performed plasma elimination studies in mice which showed that 125 I-plasma CPB remains in the circulation despite its small size. Native polyacrylamide gel electrophoresis of blood samples removed from the mice revealed that plasma CPB migrated as a high molecular weight band. Similar bands were observed in vitro when 125 I-plasma CPB was added to plasma from humans and other species. The plasma CPB-binding proteins were purified from human plasma and identified as ␣ 2 -macroglobulin (␣ 2 M) and pregnancy zone protein. Only the active enzyme bound to the two ␣-macroglobulins, and the interaction was specific for ␣ 2 M in its native conformation, but not its receptor recognized forms. The complex between human ␣ 2 M and plasma CPB dissociated during SDS-polyacrylamide gel electrophoresis and transverse urea gel electrophoresis suggesting that the interaction was noncovalent and depended on the tertiary structure of the native ␣ 2 M molecule. The catalytic activity of plasma CPB was not significantly affected by its binding to ␣ 2 M. The specific binding of plasma CPB to ␣-macroglobulins suggest that these proteins may function as a "shuttle" in vivo to modulate the clearance of plasma CPB from the circulatory system.

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“…Elution was then begun with a linear gradient of 50-150mM NaCl in 50mM Tris/HCl buffer pH 7.5, and 7.5-m/ fractions were collected. Active fractions (numbers [34][35][36][37][38][39][40][41][42], which emerged at NaCl concentrations ranging from 60 to 80mM, were combined, and dialysed against 20mM sodium phosphate buffer, pH 7.5, containing 0.14M NaCl. The dialysed fraction was then loaded to a nickel chelate column (1.2 χ 15 cm) which had been equilibrated with the same buffer.…”

Section: Resultsmentioning

confidence: 99%