Guinea Pig Plasma Murinoglobulin. Purification and Some Properties

Suzuki, Yasuyuki; Sinohara, Hyogo

doi:10.1515/bchm3.1986.367.2.579

Cited by 7 publications

(3 citation statements)

References 17 publications

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“…190 kDa belonging to the a-macroglobulin family (Esnard et al, 1985;Saito and Sinohara, 1985a, b;Suzuki and Sinohara, 1986;Lonberg-Holm et al, 1987;Schweizer et al, 1987;Braciak et al, 1988). The biosynthesis of a113 in isolated rat hepatocytes has recently been studied .…”

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Intracellular modifications of rat α₁inhibitor₃

Sjöberg

Esnard

Fries

1991

European Journal of Biochemistry

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alInhibitor3 (ai13) is a monomeric protease inhibitor of about 190 kDa which is secreted by rodent hepatocytes. We have studied intracellular modifications of this protein in [35S]methionine-labelled rat hepatocytes by pulse/ chase experiments followed by immunoprecipitation and gel electrophoresis under reducing and nonreducing conditions. Directly after the pulse, most of the unreduced a113 migrated faster than the reduced form, indicating that disulphide bridges are formed during or shortly after synthesis yielding a compact structure. With increasing chase time however, an increasing portion of the unreduced al13 migrated with a mobility lower than that of the reduced protein, half-maximal conversion occurring after about 10 min. This finding suggests that a113 undergoes a conformational change in the endoplasmic reticulum, possibly becoming more elongated. During 10 -30 min of chase, the protein acquired the capacity to undergo autolytic cleavage upon heating, a property due to the existence of an internal thiolester bond [Howard, J. B., Vermeulen Analysis by subcellular fractionation indicated that this bond is formed in the endoplasmic reticulum. Finally, we show that secreted al13 is sulphated, presumably at Tyr618.After synthesis by ribosomes attached to the endoplasmic reticulum, secretory proteins pass through the Golgi complex before being released into the medium. The Golgi complex has been shown to consist of at least three functionally different compartments (the cis, medial and trans-Golgi) through which the secretory proteins pass in succession (reviewed by Pfeffer and Rothman, 1987). Most secretory proteins are modified during their transport. The various enzymes catalysing these modifications occur in specific compartments. Therefore, the modifications and the corresponding enzyme activities may be used for the analysis of the secretory pathway. The best characterised modifications are those entailing the acquisition of covalently linked groups, such as oligosaccharides (for a review see Kornfeld and Kornfeld, 1985). It is becoming increasingly clear, however, that many secretory proteins also undergo conformational changes before their release, particularly in the endoplasmic reticulum (reviewed by Hurtley and Helenius, 1989). The molecular mechanisms underlying these processes are poorly understood.We are using isolated rat hepatocytes to study protein secretion. alInhibitor3 (a113) is one of the proteins synthesised at the highest rates in these cells. a113, also called murinoglobulin, is a rodent-specific protease inhibitor of Correspondence to M. Sjoberg,

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Intracellular modifications of rat α₁inhibitor₃

Sjöberg

Esnard

Fries

1991

European Journal of Biochemistry

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“…a-macroglobulins have been identified in a wide variety of organisms, including mammals, birds, amphibians, fish and reptiles . In addition, dimeric a-macroglobulins have been identified in the horseshoe crab (Enghild et al, 1990), octopus (Thogersen et al, 1992), crayfish (Hergenhahn et al, 1988;Stocker et al, 1991), lobster (Spycher et al, 1987) and human (Christensen et al, 1989) and monomeric a-macroglobulins have been identified in rodents, specifically the rat (Esnard et al, 1985;Enghild et al, 1989), mouse , guinea pig (Suzuki and Sinohara, 1986) and hamster . It has been suggested that a-macroglobulins, both proteinase inhibitors and complement components, have evolved from a common monomeric precursor (Sottrup-Jensen et al, 1985).…”

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Identification of monomeric α-macroglobulin proteinase inhibitors in birds, reptiles, amphibians and mammals, and purification and characterization of a monomeric α-macroglobulin proteinase inhibitor from the American bullfrog Rana catesbiana

Rubenstein

Thøgersen

Pizzo

et al. 1993

Biochemical Journal

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The alpha-macroglobulins are classified as broad-spectrum inhibitors because of their ability to entrap proteinases of different specificities and catalytic class. Tetrameric and dimeric alpha-macroglobulins have been identified in a wide variety of organisms including those as primitive as the mollusc Octopus vulgaris; however, monomeric alpha-macroglobulin proteinase inhibitors have been previously identified only in rodents. The monomeric alpha-macroglobulin proteinase inhibitors are believed to be analogous to the evolutionary precursor of the multimeric members of this family exemplified by the tetrameric human alpha 2-macroglobulin. Until now, monomeric alpha-macroglobulin proteinase inhibitors have only been identified in rodents and have therefore been considered an evolutionary anomaly. However, in this report we have utilized several sensitive assays to screen various plasmas and sera for the presence of monomeric alpha-macroglobulins, and our results suggest that monomeric alpha-macroglobulin proteinase inhibitors are present in organisms belonging to the avian, reptilian, amphibian and mammalian classes of the chordate phylum. This indicates that these proteins are more widespread than previously recognized and that their presence in rodents is not an anomaly. To demonstrate further that the identified proteins were indeed monomeric alpha-macroglobulin proteinase inhibitors, we purified the monomeric alpha-macroglobulin from the American bullfrog Rana catesbeiana. We conclude that this protein is a monomer of 180 kDa on the basis of its behaviour on (i) pore-limit gel electrophoresis, (ii) non-reducing and reducing SDS/PAGE and (iii) gel-filtration chromatography. In addition, we demonstrate that this protein is an alpha-macroglobulin proteinase inhibitor by virtue of (i) its ability to inhibit proteinases of different catalytic class, (ii) the presence of a putative internal beta-cysteinyl-gamma-glutamyl thioester and (iii) an inhibitory mechanism characterized by steric protection of the proteinase active site and by sensitivity to small primary amines. The frog monomeric alpha-macroglobulin is structurally and functionally similar to the well-characterized monomeric alpha-macroglobulin proteinase inhibitor rat alpha 1-inhibitor-3.

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“…The proteinases within such traps remain active against small substrates but are sterically hindered from access to large, macromolecular substrates. Several unusual monomeric members of the a-macroglobulin family have been described that behave like proteinase inhibitors not complement components [Saito and Sinohara, 1985;Suzuki and Sinohara, 1986;LonbergHolm et al, 1987;Rubenstein et al, 19931. These inhibitors are able to block the active site of the proteinase by some form of steric hindrance despite their inability t o form a cage around the proteinase [Enghild et al, 1989;Rubenstein et al, 19931. A domain structure has been proposed for both the human a2M subunits [Thomsen and Sottrup-Jensen, 19931 and al13 [Rubenstein et al, 19911.…”

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Binding of rat α1-inhibitor-3-methylamine to the α2-macroglobulin signaling receptor induces second messengers

1996

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Binding of receptor-recognized forms of tetrameric human alpha 2-macroglobulin (alpha 2M*) to a macrophage signaling receptor induces cAMP synthesis, increases in inositol 1,4,5-triphosphate (IP3) synthesis, and a concomitant rise in cytosolic free calcium ([Ca2+]i). The alpha 2M* signaling receptor is coupled to a pertussis-toxin insensitive G protein. Binding of alpha 2M* also occurs to the low density lipoprotein receptor-related protein/alpha 2M receptor (LRP/alpha 2MR), but this binding does not induce signal transduction. Rat alpha 1-inhibitor-3 (alpha 1I3) is a monomeric member of the alpha-macroglobulin/complement superfamily. Like alpha 2M, it can react with proteinases or methylamine which induces a conformational change causing activated alpha 1I3 to bind to LRP/alpha 2MR. We now report that alpha 1I3-methylamine binds to the macrophage alpha 2M* signaling receptor inducing a rapid rise in the synthesis of IP3 with a subsequent 1.5- to 3-fold rise in [Ca2+]i. alpha 1I3-methylamine binding to macrophages also caused a statistically significant elevation in cAMP. Native alpha 1I3, like alpha 2M, was unable to induce signal transduction. alpha 1I3 forms a complex with alpha 1-microglobulin, which has a distinct conformation from alpha 1I3 and is recognized by LRP/alpha 2MR. This complex also induces an increase in [Ca2+]i comparable to the effect of alpha 1I3-methylamine on macrophages. It is concluded that activation of alpha 1I3 by methylamine or binding of alpha 1-microglobulin causes similar conformational changes in the inhibitor, exposing the receptor recognition site for the alpha 2M* signaling receptor, as well as for LRP/alpha 2MR.

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Guinea Pig Plasma Murinoglobulin. Purification and Some Properties

Cited by 7 publications

References 17 publications

Intracellular modifications of rat α₁inhibitor₃

Intracellular modifications of rat α₁inhibitor₃

Identification of monomeric α-macroglobulin proteinase inhibitors in birds, reptiles, amphibians and mammals, and purification and characterization of a monomeric α-macroglobulin proteinase inhibitor from the American bullfrog Rana catesbiana

Binding of rat α1-inhibitor-3-methylamine to the α2-macroglobulin signaling receptor induces second messengers

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Guinea Pig Plasma Murinoglobulin. Purification and Some Properties

Cited by 7 publications

References 17 publications

Intracellular modifications of rat α1inhibitor3

Intracellular modifications of rat α1inhibitor3

Identification of monomeric α-macroglobulin proteinase inhibitors in birds, reptiles, amphibians and mammals, and purification and characterization of a monomeric α-macroglobulin proteinase inhibitor from the American bullfrog Rana catesbiana

Binding of rat α1-inhibitor-3-methylamine to the α2-macroglobulin signaling receptor induces second messengers

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Intracellular modifications of rat α₁inhibitor₃

Intracellular modifications of rat α₁inhibitor₃