STRUCTURE OF α<sub>2</sub>‐MACROGLOBULIN‐PROTEASE COMPLEXES

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

doi:10.1111/j.1749-6632.1983.tb18095.x

Cited by 23 publications

(25 citation statements)

References 8 publications

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“…The idea that bound proteinases might form a 'protein bridge' between subunits of a2M was first proposed by Krebs et al (1978) on the basis of the ability of trypsin to decrease the dissociation of a2M into subunits as observed in the ultracentrifuge. The major evidence for structures in which two proteolysed subunits were cross-linked by two bonds to an enzyme molecule comes from studies of two-dimensional electophoresis (Wang et al, 1983). 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.…”

Section: Discussion Multivalent Enzyme-a2m Complexesmentioning

confidence: 81%

“…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%

“…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). To examine further the role and nature of covalent complexes we performed kinetic experiments in which we compared the time course for formation of the various intermediates observed on non-reducing SDS/polyacrylamide-gel electrophoresis.…”

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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Section: Discussion Multivalent Enzyme-a2m Complexesmentioning

confidence: 81%

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

confidence: 99%

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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“…Previous studies have reported characteristic gel shifts representing high molecular weight, covalently cross-linked A2Msubstrate complexes [16,29]. The universal 'protease trapping' mechanism used by A2M involves a reactive thiol group that is exposed following protease-mediated cleavage of a bait region [30].…”

Section: Detection Of A2m Protein Complexes In Bloodmentioning

confidence: 99%

Prostate cancer serum biomarker discovery through proteomic analysis of alpha‐2 macroglobulin protein complexes

Burgess

Ham

Tabb

et al. 2008

Proteomics Clinical Apps

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Alpha-2 macroglobulin (A2M) functions as a universal protease inhibitor in serum and is capable of binding various cytokines and growth factors. In this study, we investigated if immunoaffinity enrichment and proteomic analysis of A2M protein complexes from human serum could improve detection of biologically relevant and novel candidate protein biomarkers in prostate cancer. Serum samples from six patients with androgen-independent, metastatic prostate cancer and six control patients without malignancy were analyzed by immunoaffinity enrichment of A2M protein complexes and MS identification of associated proteins. Known A2M substrates were reproducibly identified from patient serum in both cohorts, as well as proteins previously undetected in human serum. One example is heat shock protein 90 alpha (HSP90α), which was identified only in the serum of cancer patients in this study. Using an ELISA, the presence of HSP90α in human serum was validated on expanded test cohorts and found to exist in higher median serum concentrations in prostate cancer (n = 18) relative to control (n = 13) patients (median concentrations 50.7 versus 27.6 ng/mL, respectively, p = 0.001). Our results demonstrate the technical feasibility of this approach and support the analysis of A2M protein complexes for proteomic-based serum biomarker discovery.

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“…a2M-related proteins are present in all vertebrate species (1)(2)(3)(4). Human a2M is a tetramer of four identical 185-kDa subunits, arranged as a pair of dimers each consisting of two disulfidelinked monomers (5,6). The a2M polypeptide has a so-called bait region and an internal thiol ester bond, which account for its properties as a proteinase inhibitor.…”

mentioning

confidence: 99%

Nucleotide sequence of cDNA encoding human alpha 2-macroglobulin and assignment of the chromosomal locus.

Kan¹,

Solomon²,

Belt³

et al. 1985

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

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Six a2-macroglobulin (a2M) cDNA clones were isolated from a human liver cDNA library by using synthetic oligonucleotides as hybridization probes. One of these, pa2M1, carries a 4.6-kilobase-pair insert, which was sequenced. The insert contains the coding sequences for the mature a2M polypeptide (1451 amino acids) and for a 23-amino acid signal peptide at the NH2 terminus of the precursor proa2M. At the 3' end of the insert a poly(A) addition signal A-A-T-A-A-A and part of the poly(A) tail of the messenger RNA were found. The protein sequence deduced from the nucleotide sequence agrees with the published a2M amino acid sequence for all except three residues. The a2M locus was assigned to human chromosome 12 by Southern blot analysis with DNA from a panel of mouse/human somatic cell hybrids, using a2M cDNA as a hybridization probe.a2-Macroglobulin (a2M) is a serum glycoprotein and a major plasma proteinase inhibitor with a wide specificity. a2M-related proteins are present in all vertebrate species (1-4). Human a2M is a tetramer of four identical 185-kDa subunits, arranged as a pair of dimers each consisting of two disulfidelinked monomers (5, 6). The a2M polypeptide has a so-called bait region and an internal thiol ester bond, which account for its properties as a proteinase inhibitor. The bait region, composed of a series of target peptide bonds for plasma proteinases (7, 8) (13,14).This suggests that the conformational change, which accompanies the complex formation between a2M and proteinases and the hydrolysis of the thiol ester bond, exposes regions of the a2M molecule that are recognized by these receptors. Receptor-mediated endocytosis of proteinase-a2M complexes by macrophages and liver cells leads to clearance of the complexes from the circulation.Internal thiol ester bonds are also found in the complement proteins C3 and C4 (15), which are derived from precursor polypeptides of size similar to a2M (180-200 kDa). Their thiol ester sites are found in positions comparable to those in a2M, and the amino acid sequences of all three thiol ester sites are conserved. These observations led to the proposal that the C3, C4, and a2M genes are derived from a common ancestral gene (16). The sequences of murine and human C3 and human C4 and partial cDNA sequences of murine C4 have recently been determined (17-21). Comparison of human a2M with murine C3 revealed a 25% overall sequence homology (17,18,22

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STRUCTURE OF α₂‐MACROGLOBULIN‐PROTEASE COMPLEXES

Cited by 23 publications

References 8 publications

Kinetics of the reaction of thrombin and α2-macroglobulin

Kinetics of the reaction of thrombin and α2-macroglobulin

Prostate cancer serum biomarker discovery through proteomic analysis of alpha‐2 macroglobulin protein complexes

Nucleotide sequence of cDNA encoding human alpha 2-macroglobulin and assignment of the chromosomal locus.

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STRUCTURE OF α2‐MACROGLOBULIN‐PROTEASE COMPLEXES

Cited by 23 publications

References 8 publications

Kinetics of the reaction of thrombin and α2-macroglobulin

Kinetics of the reaction of thrombin and α2-macroglobulin

Prostate cancer serum biomarker discovery through proteomic analysis of alpha‐2 macroglobulin protein complexes

Nucleotide sequence of cDNA encoding human alpha 2-macroglobulin and assignment of the chromosomal locus.

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STRUCTURE OF α₂‐MACROGLOBULIN‐PROTEASE COMPLEXES