A Review of: “Biochemistry and Methodology of Lipids,Eds. A.R. Johnson and J.B. Davenport Wiley-Interscience,New York, 1971 x + 578 pages,ill., hard cover; $29.50”

A previous communication from this laboratory¹ as well as one from another³ described the separation of α₂-macroglobulin from swine serum. The products from both laboratories contained, in addition to α₂-macroglob-ulin, an additional macroglobulin contaminant with α₂-globulln mobility. Due to their physicochemical similarity these macroglobulins are not resolved using conventional column procedures such as ion exchange chromatography and gel filtration. Subsequent experiments have shown that immunoelectro-phoretically pure swine α₂-macroglobulln is present, in good yield (65%) in the breakthrough effluent of columns of Bio-Gel A-1.5m-Reactive Blue 2 while the contaminating macroglobulin is tightly bound. The production of highly purified swine α₂-macroglobulin utilizing this observation is the subject of the present report. The product of the separation was found to be homogeneous when subjected to Immunoelectrophoresis, at a concentration of 14–16 mg/ml, and diffused against antiswlne whole serum antibody. The production of monospecific antibody, a more stringent test for homogeneity, resulted when the purified α₂-macroglobulin was injected into rabbits. Physicochemical analyses on the purified product showed that swine and human α₂-macro-globulins are true homologs.     

Major secretory product of the mesometrial decidua in the rat,a variant of alpha-2-macroglobulin,binds insulin-like growth factor I via a protease-dependent mechanism

Decidualization-associated protein (DAP), the quantitatively major secretory product of the mesometrial decidua in the rat, is a pl variant of the liver-derived acute-phase reactant, alpha-2-macroglobulin (α₂M). α₂M, a broad spectrum protease inhibitor, has been demonstrated in the human to bind a variety of cytokines and growth factors. In humans, the quantitatively major secretory product of decidual tissue is an insulin-like growth factor (IGF) binding protein. In this study, we have therefore tested the ability of liver- and decidual-derived α₂M in the rat to bind IGF-I. α₂M purified from acute-phase plasma and DAP purified from cytosolic extracts of decidual tissue and medium from tissue incubations both bound radiolabeled IGF-I. The binding of IGF-I was principally dependent upon the coincubation of the protein with a proteinase. Therefore, it occurred during the conversion of the “slow” to the “fast” form of α₂M. Pretreatment with proteinase to produce the fast form before addition of the IGF-I reduced the binding. Binding was enhanced at a ratio protein:proteinase of 1:1. Results from gel electrophoretic analysis were consistent with the covalent linkage of IGF-1 to α₂M during the cleavage of the “bait region.” A saturable displacement by increasing concentrations of unlabeled IGF-I suggested high affinity interaction. Under conditions of demonstrated binding to purified proteins binding in acute-phase plasma, decidual tissue extracts and tissue incubation medium were associated with a high molecular weight species which was confirmed to represent α₂M and DAP, respectively. Our studies demonstrate that IGF-I may now be added to the list of regulatory peptides which α₂M may bind and that, in rat decidua, DAP may represent the functional homolog of decidual IGFBP-1 in the human and regulate growth factor function during placental development. © 1996 Wiley-Liss, Inc.     

Binding and degradation of α2-macroglobulin by cultured fibroblasts

We studied the interactions of α₂-macroglobulin, a major protease inhibitor of plasma and of serum-containing culture medium, with cultured fibroblasts. Iodinated human α₂-macroglobulin bound specifically to washed cell layers of cultured human fibroblasts. At 0–4°C, binding was saturated at a concentration of 10–20 μg/ml. At 37°C, radiolabel appered in the medium in a form soluble in 10% trichloroacetic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that ingested iodinated α₂-macroglobulin transiently forms a complex with a trypsin-like protease. Indirect immunofluorescence demonstrated α₂-macroglobulin in vacuoles of fibroblasts grown in 10% human serum or incubated with purified α₂-macroglobulin. Fibroblasts transformed by SV-40 (VA-13 cells) bound and degraded less ¹²⁵I-labeled α₂-macroglobulin than non-transformed fibroblasts and had fewer vacuoles containing α₂-macroglobulin. These observations indicate that cultured fibroblasts bind, take up by endocytosis, and degrade α₂-macroglobulin. Binding and endocytosis of α₂-macroglobulin by a cell may be a means of modulating proteases in the micro-environment of the cell and during endocytosis.     

Uptake of 125I-labelled α2-macroglobulin and albumin by human placental syncytiotrophoblast in vitro

We have investigated the binding and internalization of α₂-macroglobulin and serum albumin by human placental syncytiotrophoblast cells in vitro. The time course (obtained at 4°C) of α₂-macroglobulin binding indicated that an equilibrium was reached after 4 h. The binding of ¹²⁵I-labelled α₂-macroglobulin to syncytiotrophoblast cells was competitively reduced in the presence of excess unlabelled α₂-macroglobulin. When the concentration-dependence of binding was examined over a wide concentration range, non-linear regression analysis yielded a K_d of 6.4 nM. In the case of albumin, binding was weak and ligand dissociated from the cell surface during aqueous washing making it impractical to analyze the binding reaction. In other experiments, syncytiotrophoblast cells were incubated with ¹²⁵I-labelled α₂-macroglobulin at 37°C. Under these conditions, trypsin-resistant cell-associated radioactivity increased with time consistent with ligand internalization. ¹²⁵I-Labelled-ligand was internalized with a t_1/2 of about 5 min. After a lag period some radioactivity was released back into the incubation medium. When measured at times up to 210 min, this was found to consist of mostly TCA-precipitable material that had been lost from the cell surface. However, when the incubation was extended to 24 h, almost 15% of the initial cell-associated radioactivity was released to the extracellular medium as TCA-soluble material, consistent with a slow rate of ligand degradation. The specific binding of ⁶⁵Zn-labelled α₂M was similar to that of the ¹²⁵I-labelled ligand and trypsin-resistance measurements provided evidence of α₂M-mediated ⁶⁵Zn uptake. These results support a role for syncytiotrophoblast in the metabolism of α₂-macroglobulin during pregnancy and are also consistent with a role for α₂-macroglobulin in the maternal-fetal transport of zinc. J. Cell. Biochem. 68:427–435, 1998. © 1998 Wiley-Liss, Inc.     

CULTIVATION OF MAMMALIAN CELLS IN DEFINED MEDIA WITH PROTEIN AND NONPROTEIN SUPPLEMENTS

An improved chemically defined basal medium (CMRL-1415) has been used to advantage in studying the effects on trypsinized, newly explanted mouse embryo cells of certain glycoproteins from plasma and serum, certain nonprotein macromolecules, and various combinations of these, in stationary cultures. When protein and nonprotein fractions were separated from fetal calf serum, the entire growth activity was found to be associated with the protein. When 100 mg % of dialyzed, freeze-dried, supernatant solution of Cohn's fraction V (method 6) from human plasma was used as a supplement for CMRL-1415, there was considerable improvement in the cultures; and seromucoid prepared from calf serum had a similar effect. Supernatant V was further fractionated by gel filtration to give a threefold concentration of growth activity in a single, highly purified α₁-acid glycoprotein (orosomucoid). Starch gel electrophoresis of horse serum that was used to supplement the basal medium revealed a decrease of both α₁-acid glycoprotein and α₂-macroglobulin during the cultivation of mouse embryo cells. When horse serum was fractionated on DEAE-cellulose columns, the only fraction that showed growth activity was a slow α₂-globulin. When the α₂-macroglobulin of Schultze was prepared from horse serum by salt precipitation, it was equally effective. When the α₂-macroglobulin from horse serum was tested (at 100 mg %) in combination with α₁-acid glycoprotein from Supernatant V, seromucoid from calf serum, or unfractionated Supernatant V, the growth response was greatly in excess of that produced by any of these supplements tested separately. The α₂-macroglobulin from horse serum could be replaced by certain nonprotein macromolecules (e.g., dextran or Ficoll). Thus, dextran (mol. wt. 100,000 to 200,000) had no visible effect on the cells when used alone at 0.1 or 1%. But when these levels of dextran were used in combination with low molecular weight glycoproteins (e.g., unfractionated Supernatant V at 100 mg %), the cultures remained active and healthy for unusually long periods.     

Separation of the molecular forms of the insulin-like growth factor (IGF)—Binding proteins by affinity chromatography

Association of IGFBP-1, IGFBP-2 and IGFBP-3 with other proteins in human serum and placental cell membranes was investigated using affinity chromatography matrix with immobilized antibodies. Circulating IGFBP-1 was found to be predominantly bound to α₂-macroglobulin and not in the binary complex with its ligand, IGFBP-2 complexes and/or polymers were detected, which was not acknowledged before, and IGFBP-3 molecular forms were differentiated into those that form binary/ternary complexes and those that form stable associations with other serum proteins. As for placental membranes, both IGFBP-1 dimers and high molecular mass IGFBP-1 associations, most probably with α₂-macroglobulin, were recognized and resolved.     

Purification and properties of a fibrinogenase from the venom of Naja nigricollis

A fibrinogenolytic proteinase from the venom of Naja nigricollis was purified by chromatography on Bio-Rex 70 and Phenyl-Sepharose. The purified enzyme, designated proteinase F1, was homogeneous by the criterion of SDS-polyacrylamide gel electrophoresis, and consisted of a single chain with a molecular weight of 58 000. Purified proteinase F1 had approximately 15-fold more proteinase activity than the crude venom, based on its ability to inactive α₂-macroglobulin. The enzyme acted on only the Aα-chain of fibrinogen and left the Bβ- and γ-chains intact. The pH optimum for this fibrinogenolytic activity was in the range of pH 8 to 10. In addition to its activity on fibrinogen, proteinase F1 was active on α₂-macroglobulin and fibronectin, but did not degrade casein, hemoglobin or bovine serum albumin. The enzyme was not inhibited by inhibitors of serine proteinases, cysteine proteinases or acid proteinases, but only by the metalloproteinase inhibitor, EDTA. The inhibition by EDTA could be prevented by Zn²⁺, but not by Ca²⁺ or Mg²⁺.     

A monoclonal antibody to human insulin receptor

A murine hybridoma secreting antibody against human insulin receptor was produced by fusing FO myeloma cells with spleen and lymph node cells from a mouse that had been immunized with insulin receptor purified from human placenta. The secreted antibody was an IgG₁ (κ), designated αIR-1. Like the previously described rabbit polyclonal antibody, αIR-1 did not inhibit insulin binding. It specifically immunoprecipitated ¹²⁵I-insulin-receptor complexes as well as unoccupied receptor previously labeled directly with lactoperoxidase. Thus, αIR-1 interacts with the receptor at a site distinct from the insulin binding site. Unlike previously described anti-insulin receptor antibodies, αIR-1 exhibits strong tissue and species specificity.     

α2-Macroglobulin as a β-Amyloid Peptide-Binding Plasma Protein

Abstract: The β-amyloid peptide (Aβ) is a normal proteolytic processing product of the amyloid precursor protein, which is constitutively expressed by many, if not most, cells. For reasons that are still unclear, Aβ is deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease (AD). The factor(s) responsible for the clearance of soluble Aβ from biological fluids or tissues are poorly understood. We now report that human α₂-macroglobulin (α₂M), a major circulating endoproteinase inhibitor, which has recently been shown to be present in senile plaques in AD, binds ¹²⁵I-Aβ_(1–42) with high affinity (apparent dissociation constant of 3.8 × 10^?10M). Approximately 1 mol of Aβ is bound per mole of α₂M. Both native and methylamine-activated α₂M bind ¹²⁵I-Aβ_(1–42). The binding of ¹²⁵I-Aβ_(1–42) to α₂M is enhanced by micromolar concentrations of Zn²⁺ (but not Ca²⁺) and is inhibited by noniodinated Aβ_(1–42) and Aβ_(1–40) but not by the reverse peptide Aβ_(40-1) or the cytokines interleukin 1β or interleukin 2. α₁-Antichymotrypsin, another plaque-associated protein, inhibits both the binding of ¹²⁵I-Aβ_(1–42) to α₂M as well as the degradation of ¹²⁵I-Aβ_(1–42) by proteinase-activated α₂M. Moreover, the binding of ¹²⁵I-Aβ_(1–42) to α₂M protects the peptide from proteolysis by exogenous trypsin. These data suggest that α₂M may function as a carrier protein for Aβ and could serve to either facilitate or impede clearance of Aβ from tissues such as the brain.     

Separation of anti-histone antibodies from nonimmune histone-precipitating serum proteins, predominantly alpha2-macroglobulin.

Calf thymus histories formed precipitates with human, rabbit, guinea pig, mouse, goat, or sheep sera regardless of whether the subjects were immunized with histones. Their order of activity in precipitation was: H4 >H3 >H2a >H2b ? H1. Precipitation occurred with more than one serum protein fraction, but not with purified IgG, IgM, or crystallized albumin. The major nonimmune precipitant had the size and charge properties of α2-macroglobulin. Serological analysis with anti-α2-macroglobulin antiserum confirmed the presence of this protein in the precipitating fractions, and its selective depletion from histone-precipitated serum. Specific anti-histone antibodies were separated from the nonimmune precipitants by DEAE-cellulose chromatography.     

Inhibition of Human Pepsin and Gastricsin by α2-Macroglobulin

The inhibitory effects of human α₂-macroglobulin (α₂-M), a major plasma proteinase inhibitor, on human pepsin and gastricsin were investigated. The activities of pepsin and gastricsin towards a protein substrate (reduced and carboxymethylated ribonuclease A) were significantly inhibited by α₂-M at pH 5.5, whereas those towards a peptide substrate (oxidized insulin B-chain) were scarcely inhibited. Under these conditions at pH 5.5, pepsin and gastricsin cleaved α₂-M mainly at the His⁶⁹⁴-Ala⁶⁹⁵ bond and Leu⁶⁹⁷-Val⁶⁹⁸ bond, respectively, in the bait regions sequence of α₂-M. The conformation of α₂-M was also shown to be markedly altered upon inhibition of these enzymes as examined by native polyacrylamide gel electrophoresis and electron microscopy. These results show the entrapment and concomitant inhibition of those proteinases by α₂-M.     

Molecular feature of the nerve growth factor in human serum

High molecular weight binding components which bind [¹²⁵I] mouse β nerve growth factor exist in human serum. The binding of β nerve growth factor to the serum components was inhibited at alkaline condition. After gel filtration of human serum on a Sephadex G-150 column at neutral condition, the nerve growth factor-like immunoreactivity was observed in only one peak, differing from the high molecular weight serum components. However, at alkaline condition two peaks with nerve growth factor-like immunoreactivity appeared; one was almost at the position observed at neutral pH, and the other was a new peak eluted approximately to the column volume. these results suggest that there are at least two nerve growth factor-like molecules in human serum and most of the nerve growth factor in the serum exists in a complex form associated with serum components with high molecular weight.     

Purification and properties of a novel recombinant human hybrid interferon, σ-4 α2/α1

The human interferon (huIFN) σ-4 α2_5–62/α1_64–166 is a genetically engineered hybrid that consists of residues 5–62 of huIFN α2 and residues 64–166 of huIFN α1. This variant contains four cysteine residues at positions 29, 86, 99 and 139, but does not contain the cysteine at position 1 that is characteristic of naturally occurring huIFN α subtypes. This novel recombinant hybrid was purified fromEscherichia coli to greater than 95% homogeneity. The purification was based on ethanol extraction of a trichloroacetic acid precipitate and Matrex Gel Blue A chromatography followed by either a selective precipitation or DEAE-Sepharose chromatography. The purified protein that was treated with 2-mercaptoethanol exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 17 800 and 17 100, both of which exhibited antiviral activity. Electrophoresis performed without prior reduction with 2-mercaptoethanol indicated only a minor extent of intermolecular disulfide bonding. The purified protein exhibited a high specific antiviral activity of 7·10⁷ units/mg when assayed on human fibroblast cells and, in distinction to the parental huIFN α2, it also demonstrated antiviral activity on murine L929 cells. The level of antiproliferative activity of huIFN δ-4 α2_5–62/α1_64–166 on various cell lines of different histological origin appeared to be more comparable to that of huIFN α1 than huIFN α2. The data suggest that huIFN δ-4 α2_5–62/α1_64–166 hybrid may be a useful tool for understanding huIFN structure-function relations.     

Previously Undescribed Fibrinolysis Inhibitor in Human Blood

HUMAN serum contains several protease inhibitors, one of which is identified as α₁-globulin (α₁-antitrypsin) and another as α₂-macroglobulin. Serum also contains inhibitors against the activation of plasminogen, which may differ from the protease inhibitors. Thus, inhibition of plasminogen activation by urokinase is selectively increased during the last trimester of pregnancy^1,2. Likewise, increased inhibition of streptokinase-activated human euglobulin or of a tissue plasminogen activator was noticed in some groups of patients with thrombotic disease^3,4. This observation is of particular interest because the tissue activator, which is present in venous endothelium⁵, is believed to assist in the removal of fibrinous deposits caused by tissue injury⁶.     

Bumetanide-sensitive Na+ /K+ /Cl− transporter is stimulated by phorbol ester and different mitogens in quiescent human skin fibroblasts

In this study we investigated the correlation between the mitogenic effect and stimulation of Rb + (K + ) fluxes in human skin fibroblasts treated by purified growth factors. Both K+ transporters, bumetanide-sensitive and ouabain-sensitive, are stimulated 2-3-fold after addition of either fetal calf serum or purified recombinant growth factors to quiescent G₀/G₁ human skin fibroblasts. Three groups of mitogens were compared: (i) the phorbol ester 2-O-tetradecanoyl-phorbol-13-acetate (TPA); (ii) growth factors that stimulate inositol phosphate hydrolysis and subsequently activate protein kinase C—fibroblast growth factor (FGF), platelet derived growth factor (PDGF), and α-thrombin; and (iii) growth factors that do not activate kinase C—insulin-like growth factor-1 (IGF-1), and transforming like growth-factor-α (TGF-α). The three groups of mitogens stimulated human skin fibroblasts proliferation and Rb+ influxes in a similar dose-dependent fashion. The results indicate that both the bumetanide-sensitive and the ouabain-sensitive Rb+ fluxes are stimulated by protein kinase C-dependent and by the protein kinase C-independent pathways of the mitogenic signal.     

Structural changes of alpha2- and ovomacroglobulins

The plasma α₂-macroglobulin and its egg white homologue ovomacroglobulin were purified from several different species and their structure before and after the reaction with proteinases studied by electron microscopy. The negatively stained specimens showed either a ringlike structure or a flowerlike one before the reaction with proteinses, but their structures changed into open rectangular ones after the reaction. The translational frictional ratio f/f ₀ of human α2-macroglobulin and crocodilian ovomacroglobulin given in the literature is between 1.5 and 1.6 before and after the reaction with proteinases. The value reflects asymmetry due not to a high axial ratio, but rather to an openness of the structure resulting in a partially free draining character of the molecules. The computational method developed by Bloomfield and his co-workers based on the formalism of Kirkwood is used to calculate the frictional ratio of several models constructed from small spheres. The overall shape of the models is derived from electron micrographs. Although the degree of hydration is an unknown parameter in the calculation, reasonable agreement is obtained between the experimental values of f/f ₀ and the calculated ones. Combination of electron microscopic and hydrodynamic methods would be fruitful in the structural study of giant proteins such as α₂-macroglobulin.     

Purification of ctp: cholinephosphate cytidylyl-transferase from pea stems

CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) was purified from pea (Pisum sativum) stems. The purification involved ammonium sulphate fractionation, ion exchange chromatography, removal of proteases with α₂-macroglobulin and gel filtration. The purified enzyme had K_m values for phosphorylcholine and CTP of 2.1 mM and 0.55 mM respectively. It was found to have a pH optimum of 7.5, a requirement for Mg²⁺ and an M_r of 56000. It could not utilize phosphorylethanolamine and its activity was not stimulated by added phospholipids.     

Cross-species Binding Analyses of Mouse and Human Neonatal Fc Receptor Show Dramatic Differences in Immunoglobulin G and Albumin Binding

The neonatal Fc receptor (FcRn) regulates the serum half-life of both IgG and albumin through a pH-dependent mechanism that involves salvage from intracellular degradation. Therapeutics and diagnostics built on IgG, Fc, and albumin fusions are frequently evaluated in rodents regarding biodistribution and pharmacokinetics. Thus, it is important to address cross-species ligand reactivity with FcRn, because in vivo testing of such molecules is done in the presence of competing murine ligands, both in wild type (WT) and human FcRn (hFcRn) transgenic mice. Here, binding studies were performed in vitro using enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant soluble forms of human (shFcRn^WT) and mouse (smFcRn^WT) receptors. No binding of albumin from either species was observed at physiological pH to either receptor. At acidic pH, a 100-fold difference in binding affinity was observed. Specifically, smFcRn^WT bound human serum albumin with a K_D of ∼90 μm, whereas shFcRn^WT bound mouse serum albumin with a K_D of 0.8 μm. shFcRn^WT ignored mouse IgG1, and smFcRn^WT bound strongly to human IgG1. The latter pair also interacted at physiological pH with calculated affinity in the micromolar range. In all cases, binding of albumin and IgG from either species to both receptors were additive. Cross-species albumin binding differences could partly be explained by non-conserved amino acids found within the α2-domain of the receptor. Such distinct cross-species FcRn binding differences must be taken into consideration when IgG- and albumin-based therapeutics and diagnostics are evaluated in rodents for their pharmacokinetics.     

Interaction between human pancreatic elastase and plasma protease inhibitors

1 ml of human serum inhibits about 0.9 mg of purified human pancreatic elastase owing to complexation with alpha 1-antitrypsin and alpha 2-macroglobulin. On addition to serum, elastase is preferentially bound by alpha 2-macroglobulin. The complexes between elastase and alpha 1-antitrypsin and alpha 2-macroglobulin, respectively, migrate as alpha 2-globulin on agarose gel electrophoresis. Elastase bound by alpha 1-antitrypsin is precipitated by antibodies against enzyme as well as inhibitor, while the alpha 2-macroglobulin-bound elastase is only precipitated by antibodies against the inhibitor. The molar combining ratio for elastase/alpha 1-antitrypsin is 1:1 and for elastase/alpha 2-macroglobulin 2:1. The elastase bound by alpha 2-macroglobulin retains its activity against low molecular weight substrates, while that bound by alpha 1-antitrypsin is enzymologically inactive.