Biomedical subjects

J J Enghild

Publications and source records attributed to J J Enghild.

At least 37 records · Page 2Linked to original sources

PubMed1997

The paradigm that all oxygen-respiring eukaryotes have cytosolic CuZn-superoxide dismutase and that Mn-superoxide dismutase is localized to the mitochondria does not apply to a large group of marine arthropods.

The enzyme superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide radical, is present in the cytosol and mitochondria of all oxygen-respiring eukaryotes. The cytosolic form contains copper and zinc (CuZnSOD), whereas the mitochondrial form contains manganese (MnSOD). The latter protein is synthesized in the cytosol as a MnSOD precursor, containing an N-terminal mitochondrial-targeting sequence. CuZnSOD is sensitive toward cyanide (CN) and hydrogen peroxide (H2O2), but MnSOD is not. Assays for SOD activity in cytosol from the hepatopancreas of the blue crab, Callinectes sapidus, showed the presence of a CN/H2O2-insensitive form of SOD. No CN/H2O2-sensitive CuZnSOD was found. This unexpected phenomenon was shown to occur in all decapod crustacea (crabs, lobsters, shrimp) examined. The cytosolic and mitochondrial SODs of C. sapidus were purified by means of ion-exchange, size-exclusion, and reverse-phase HPLC. The cytosolic SOD is a homodimeric protein, which exists in a monomer-dimer equilibrium (24 kDa left and right arrow 48 kDa). The protein contains approximately 1 Mn per subunit. No copper or zinc is present. Amino acid sequence analysis identified the novel cytosolic SOD as a MnSOD precursor with an abnormal mitochondrial-targeting sequence. The mitochondrial SOD of C. sapidus is similar to the MnSOD found in other eukaryotes. N-Terminal amino sequences of mitochondrial and cytosolic blue crab MnSOD differ in several positions. The MnSODs are thus encoded for by two different genes. The paradigm that all eukaryotes contain intracellular CuZnSOD and that MnSOD occurs exclusively in the mitochondria appears not to apply to a large group of marine arthropods.

Amino Acid Sequence↗

PubMed1997

Assignment of a single disulphide bridge in human alpha2-antiplasmin: implications for the structural and functional properties.

Human alpha2-antiplasmin (alpha2AP) is a serpin involved in the regulation of blood coagulation. Most serpins, unlike smaller serine proteinase inhibitors, do not contain disulphide bridges. alpha2AP is an exception from this generalization and has previously been shown to contain four Cys residues organized into two disulphide bridges [Lijnen, Holmes, van Hoef, Wiman, Rodriguez and Collen (1987) Eur. J. Biochem. 166, 565-574]. However, we found that alpha2AP incorporates iodo[14C]acetic acid, suggesting that the protein contains reactive thiol groups. This observation prompted a re-examination of the state of the thiol groups, which revealed (i) a disulphide bridge between Cys43 and Cys116, (ii) that Cys76 is bound to a cysteinyl-glycine dipeptide, and (iii) and Cys125 exists as either a free thiol or in a mixed disulphide with another Cys residue. The disulphide identified between Cys43 and Cys116 appears to be conserved in orthologous proteins since the homologous Cys residues form disulphide bonds in bovine and possibly mouse alpha2AP. The conservation of this disulphide bridge suggests that it is important for functional aspects of alpha2AP. However, the structural and functional analysis described in this study does not support this conclusion.

Animals↗

PubMed1997

Prothrombin, albumin and immunoglobulin A form covalent complexes with alpha1-microglobulin in human plasma.

Molecules containing the 33-kDa plasma protein alpha1-microglobulin were isolated from human plasma by anti-(alpha1-microglobulin) affinity chromatography. Five major bands could be seen after electrophoretic separation of the alpha1-microglobulin-containing proteins under native conditions. Immunoblotting demonstrated alpha1-microglobulin in all five bands. Two of these have been described previously: free alpha1-microglobulin and alpha1-microglobulin complexed with IgA (IgA x alpha1-microglobulin). The other three bands were identified as prothrombin alpha1-microglobulin, albumin x alpha1-microglobulin and dimeric alpha1-microglobulin. Prothrombin x alpha1-microglobulin were 1:2 and 1:1 complexes which carried approximately 1% of total alpha1-microglobulin, had molecular masses of about 145 kDa and 110 kDa upon SDS/PAGE and dissociated completely to free alpha1-microglobulin and prothrombin (72 kDa) when reducing agents were added, suggesting that the complexes were stabilized by disulfide bonds. The alpha1-microglobulin molecules did not inhibit cleavage of prothrombin by factor Xa and were bound to the peptides which were released upon activation of prothrombin. Albumin x alpha1-microglobulin, corresponding to 7% of total plasma alpha1-microglobulin, was a mixture between 1:1 and 1:2 complexes, with masses upon SDS/PAGE of approximately 100 kDa and 135 kDa, respectively. Both these complexes dissociated only partially to free alpha1-microglobulin and albumin when reducing agents were added. The albumin x alpha1-microglobulin complexes carried a yellow-brown chromophore similar to free alpha1-microglobulin. The complex-binding to alpha1-microglobulin did not block the fatty-acid-binding ability of albumin. The plasma concentrations of albumin x alpha1-microglobulin and prothrombin x alpha1-microglobulin were estimated to 5.2 mg/l and 1.1 mg/l, respectively.

Alpha-Globulins↗

PubMed1997

Evidence for a novel O-linked sialylated trisaccharide on Ser-248 of human plasminogen 2.

Human plasminogen, the inactive precursor of plasmin, exists in two major glycoforms. Plasminogen 1 contains an N-linked oligosaccharide at Asn-289 and an O-linked oligosaccharide at Thr-345. Plasminogen 2 is known to contain only an O-linked oligosaccharide at Thr-345. However, plasminogen 2 displays a further well documented microheterogeneity dependent on the N-acetylneuraminic acid content, which has functional consequences with regard to activation of plasminogen. The proposed structure and number of known oligosaccharide linkages in plasminogen 2 is insufficient to account for this microheterogeneity. In the present study, a combination of trypsin digestion, lectin affinity chromatography, Edman degradation amino acid sequence analysis, carbohydrate composition analysis, and mass spectrometry revealed the existence of a novel site for O-linked glycosylation on plasminogen 2 at Ser-248. Direct evidence for the structure of the carbohydrate was obtained from a combination of lectin affinity chromatography, desialylation experiments, and mass spectrometry analysis. These findings provide a structural basis for some of the observed microheterogeneity, and have implications with regard to the known functional consequences of the extent of sialylation of plasminogen.

Amino Acid Sequence↗

PubMed1997

The potential role of alpha 2-macroglobulin in the control of cysteine proteinases (gingipains) from Porphyromonas gingivalis.

Porphyromonas gingivalis is closely associated with the development of some forms of periodontitis. The major cysteine proteinases released by this bacterium hydrolyze peptide bonds only after arginyl (gingipain R) or lysyl residues (gingipain K). No target protein inhibitors have been identified for either enzyme, leading us to investigate their inhibition by human plasma alpha 2-macroglobulin (alpha 2M). Both 50- and 95 kDa gingipain R were efficiently inhibited by alpha 2M, whereas the catalytic activity of gingipain K could not be eliminated. All 3 enzymes were, however, inhibited by a homologous macroglobulin from rat plasma, alpha 1-inhibitor-3 (alpha 1I3). alpha-Macroglobulins must be cleaved in the so-called "bait region" in order to inhibit proteinases by a mechanism involving physical entrapment of the enzyme. A comparison of the amino acid sequences of the 2 macroglobulins indicates that the lack of lysyl residues within the bait region of alpha 2M protects Lys-specific proteinases from being trapped. On this basis, other highly specific proteinases might also not be inhibited by alpha 2M, possibly explaining the inability of the inhibitor to control proteolytic activity in some bacterially induced inflammatory states, despite its abundance (2-5 mg/ml) in vascular fluids.

Adhesins, Bacterial↗

PubMed1997

Mouse extracellular superoxide dismutase: primary structure, tissue-specific gene expression, chromosomal localization, and lung in situ hybridization.

Extracellular superoxide dismutase (EC-SOD) is the major extracellular antioxidant enzyme. We have determined the primary structure of mouse EC-SOD by characterization of complementary DNA (cDNA) clones and by amino-acid sequence analysis of purified protein. cDNA sequence analysis indicates that mouse EC-SOD is synthesized as a 251-amino-acid precursor protein with a predicted molecular weight of 27,400 D. Amino-terminal micro sequence analysis of purified mature mouse lung EC-SOD demonstrated the sequence to begin with SSFDLADRLDPV-. These results indicate that EC-SOD as initially synthesized contains a 24-amino-acid precursor peptide, and that the mature protein is 227 amino acids in length. Computer algorithms that predict the most likely site of cotranslational signal peptidase cleavage suggest that processing will occur between amino acids 18 and 19 or 20 and 21, which implies that EC-SOD may be initially synthesized as a pre-pro-protein. Like human EC-SOD, mature mouse EC-SOD is glycosylated. The full-length mouse EC-SOD cDNA is 1,834 base pairs long and is 82% (79% for protein) identical to rat EC-SOD, but only 60% (60% for protein) identical to human EC-SOD. The mouse EC-SOD gene locus (Sod3) was mapped by interspecific backcross haplotype analysis as being 0.9 +/- 0.9 centimorgans distal to the Qdpr locus on mouse Chromosome 5, a position suggesting that the human homologue of EC-SOD will map close to the human QDPR locus (4p15.3). Of nine tissues examined by Northern blot analysis, those of the kidney and lung are by far the major tissues that express EC-SOD messenger RNA. Using in situ hybridization in the mouse lung, we demonstrate EC-SOD gene expression to be highly localized to alveolar Type II epithelial cells. These data suggest that alveolar Type II cells play a central role in mediating EC-SOD antioxidant function in the lung.

Amino Acid Sequence↗

PubMed1997

Familial subepithelial corneal amyloidosis--a lactoferrin-related amyloidosis.

PURPOSE: To isolate the protein that collects in increased amounts beneath the corneal epithelium in familial subepithelial corneal amyloidosis (FSCA), also known as gelatinous droplike corneal dystrophy, and to identify it by N-terminal amino acid sequencing. METHODS: Peptides resulting from pepsin digestion of a unique protein isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from frozen tissue from two corneas with FSCA were purified by high-pressure liquid chromatography followed by protein sequence analysis. The protein was identified by amino acid sequencing, Western blotting, and immunohistochemistry. RESULTS: A protein was identified in two corneas with FSCA that was not present in normal corneas or in corneas with other disorders. The amino acid sequences of two peptides derived from this protein were identical to portions of lactoferrin. The unique protein reacted with rabbit antihuman lactoferrin after Western blotting. The presence of lactoferrin in the amyloid within affected corneas was confirmed using the immunoperoxidase method on formalin-fixed, paraffin-embedded tissue sections and lactoferrin antiserum. CONCLUSIONS: Corneal tissue with FSCA contains lactoferrin, and this is the first form of amyloidosis found to be associated with this protein. Because lactoferrin is a product of lacrimal glands, the corneal lactoferrin may be derived from the tears. Because the gene for lactoferrin is on chromosome 3 (3q21-q23), this locus is a potential site for the FSCA gene.

Adolescent↗

PubMed1996

Structural and functional analysis of the spontaneous re-formation of the thiol ester bond in human alpha 2-macroglobulin, rat alpha 1-inhibitor-3 and chemically modified derivatives.

The alpha-macroglobulins are proteinase inhibitors that form part of a superfamily along with components of the complement system. Internal beta-cysteinyl-gamma-glutamyl thiol ester bonds are an important structural feature of most alpha-macroglobulins and several complement components. We have studied the reversibility of thiol ester cleavage caused by NH3 or CH3NH2 in tetrameric human alpha 2-macroglobulin (alpha 2M) and monomeric rat alpha 1-inhibitor-3 (alpha 1I3). When employing NH3 as the nucleophile, the thiol ester in alpha 1I3 re-formed spontaneously at room temperature after gel filtration to remove excess nucleophile, and an active proteinase inhibitor was regained. When CH3NH2 was employed as the nucleophile, thiol ester reversibility was more energy-demanding. With either nucleophile, alpha 2M once inactivated did not regain proteinase-inhibitory capacity at room temperature. At elevated temperatures, however, the reaction between alpha 2M and NH3 or CH3NH2 was reversible and the inhibitory capacity could be recovered. Modification of the cysteinyl groups from the thiol ester prevented its re-formation but did not prevent the heat-induced retrieval of inhibitory capacity, suggesting that conformational features rather than the thiol ester are essential for alpha 2M to function as an inhibitor. As demonstrated by non-denaturing PAGE, the conformation of native alpha 2M is restored when the proteinase-inhibitory capacity is recovered.

Acute-Phase Proteins↗

PubMed1996

Human extracellular superoxide dismutase is a tetramer composed of two disulphide-linked dimers: a simplified, high-yield purification of extracellular superoxide dismutase.

Studies examining the biochemical characteristics and pharmacological properties of extracellular superoxide dismutase (EC SOD) have been severely limited because of difficulties in purifying the enzyme. Recently EC SOD was found to exist in high concentrations in the arteries of most mammals examined and it is the predominant form of SOD activity in many arteries. We now describe a three-step, high-yield protocol for the purification of EC SOD from human aorta. In the first step, the high affinity of EC SOD for heparin is utilized to obtain a fraction in which EC SOD constitutes roughly 13% of the total protein compared with only 0.3% of that of the starting material. In addition, over 80% of the original EC SOD activity present in the aortic homogenate was retained after the first step of purification. EC SOD was further purified using a combination of cation- and anion-exchange chromatography. The overall yield of EC SOD from this purification procedure was 46%, with over 4 mg of EC SOD obtained from 230 g of aorta. Purified EC SOD was found to exist predominantly as a homotetramer composed of two disulphide-linked dimers. However, EC SOD was also found to form larger multimers when analysed by native PAGE. It was shown by urea denaturation that the formation of multimers increased the thermodynamic stability of the protein. Limited proteolysis of EC SOD suggested that there is one interchain disulphide bond covalently linking two subunits. This disulphide bond involves cysteine-219 and appears to link the heparin-binding domains of the two subunits.

Amino Acid Sequence↗

PubMed1996

Degradation of interleukin 1beta by matrix metalloproteinases.

Matrix metalloproteinases (MMPs) and interleukin 1 (IL-1) are implicated in inflammation and tissue destruction, where IL-1 is a potent stimulator of connective tissue cells to produce the extracellular matrix-degrading MMPs. Here, we report that IL-1beta, but not IL-1alpha, is degraded by MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), and MMP-9 (gelatinase B). This degradation was effectively blocked by tissue inhibitor of metalloproteinases (TIMP)-1. When IL-1beta was treated with MMPs it lost the ability to enhance the synthesis of prostaglandin E2 and pro-MMP-3 in human fibroblasts. The primary cleavage site of IL-1beta by MMP-2 was identified at the Glu25-Leu26 bond. These results suggest that IL-1beta stimulates connective tissue cells to produce MMPs, but activated MMPs in turn negatively regulate the activity of IL-1beta.

Amino Acid Sequence↗

PubMed1996

Activated human plasma carboxypeptidase B is retained in the blood by binding to alpha2-macroglobulin and pregnancy zone protein.

A 66-kDa glycosylated carboxypeptidase, plasma pro-carboxypeptidase 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 COOH-terminal 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 125I-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 125I-plasma CPB was added to plasma from humans and other species. The plasma CPB-binding proteins were purified from human plasma and identified as alpha2-macroglobulin (alpha2M) and pregnancy zone protein. Only the active enzyme bound to the two alpha-macroglobulins, and the interaction was specific for alpha2M in its native conformation, but not its receptor recognized forms. The complex between human alpha2M 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 alpha2M molecule. The catalytic activity of plasma CPB was not significantly affected by its binding to alpha2M. The specific binding of plasma CPB to alpha-macroglobulins suggest that these proteins may function as a "shuttle" in vivo to modulate the clearance of plasma CPB from the circulatory system.

Carboxypeptidase B↗

PubMed1996

Hamster antithrombin III: purification, characterization and acute phase response.

Antithrombin III was purified to homogeneity from hamster plasma by affinity chromatography on heparin-agrose, ion-exchange chromatography on Mono Q and size-exclusion chromatography on TSK G3000SWG column with 50% yield. The molecular mass of hamster antithrombin III was estimated at 62.5 kDa and the absorption coefficient (A280 nm 1%, 1 cm) at 6.48 (in 0.1 M sodium phosphate pH 7.0). Several isoforms of the inhibitor were detected with the pI in range of 4.95-5.25. The protein contains all residues characteristic for complex-type carbohydrate chains. The N-terminal amino acid sequence shows 84% of identity to mouse and 76% to human analogue. The hamster antithrombin III gives low immunological cross-reactivity with antibodies to human antithrombin III. Initiation of the acute phase response only slightly affected the plasma concentration of inhibitor (+/- 10% within 72-h period). The kinetic data suggest high efficiency in bovine and human thrombin inhibition. In summary, the study shows only similarities between hamster and other mammal antithrombins.

Acute-Phase Reaction↗

PubMed1996

Huntingtin and DRPLA proteins selectively interact with the enzyme GAPDH.

At least five adult-onset neurodegenerative diseases, including Huntingtin disease (HD), and dentatorubral-pallidoluysian atrophy (DRPLA) are produced by genes containing a variably increased CAG repeat within the coding region. The size range of the repeats is similar in all diseases; unaffected individuals have fewer than 30 CAG repeats, whereas affected patients usually have more than 40 repeats. The size of the inherited CAG repeat correlates with the severity and age of disease onset. The CAG triplet repeat produces a polyglutamine domain in the expressed proteins. All of these diseases are inherited in a dominant fashion, and a pathologic gain of function in gene carriers has been proposed. We sought to identify proteins in the brain that selectively interact with polyglutamine-domain proteins, hypothesizing that the polyglutamine domain may determine protein-protein interactions.

Adult↗

PubMed1995

Biosynthesis of bikunin proteins in the human carcinoma cell line HepG2 and in primary human hepatocytes. Polypeptide assembly by glycosaminoglycan.

In this report we describe a series of experiments designed to probe the biosynthesis of the bikunin proteins. The bikunin proteins are serine proteinase inhibitors found in high concentrations in human plasma. The proteins are composed of two or three polypeptide chains assembled by a newly identified carbohydrate mediated covalent inter-chain "Protein-Glycosaminoglycan-Protein" (PGP) cross-link (Enghild, J. J., Salvesen, G., Hefta, S. A., Thøgersen, I. B., Rutherfurd, S., and Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751). In this study we show that transformed hepatocyte cell lines, exemplified by HepG2 cells, have lost the ability to produce these proteins. In contrast, primary human hepatocytes produce bikunin proteins identical to the proteins identified in human plasma. Pulse-chase analysis demonstrate that the PGP-mediated cross-linking of the polypeptide chains occurs late in the secretary pathway. Moreover, the mechanism responsible for the formation of the PGP cross-link is divided in two steps involving a proteolytic cleavage followed by carbohydrate attachment. The results indicate that normal hepatocytes contain the biosynthetic machinery required for correct synthesis and processing. However, transformed cell lines are defective in several aspects of bikunin biosynthesis precluding such systems from being used as relevant in vitro models.

Alpha-Globulins↗

PubMed1995

The effect of residue 1106 on the thioester-mediated covalent binding reaction of human complement protein C4 and the monomeric rat alpha-macroglobulin alpha 1 I3.

The histidine at position 1106 of the C4B isotype of human complement is involved in catalyzing the covalent binding of the thioester to glycerol and water. By replacing the histidine with other residues, it was found that tyrosine is also capable of mediating the reaction. We propose that they act as nucleophiles by first attacking the thioester, upon activation, to form acyl intermediates, which subsequently react with the hydroxyl groups of glycerol or water. The monomeric alpha-macroglobulin, alpha 1I3 of the rat, was also studied. Unlike alpha 2-macroglobulin, which is a tetramer, alpha 1I3 has binding properties similar to those of C4A.

Amino Acid Sequence↗

PubMed1995

Sodium dodecyl sulfate-stable complexes between serpins and active or inactive proteinases contain the region COOH-terminal to the reactive site loop.

Recently inhibitors of the serpin family were shown to form complexes with dichloroisocoumarine (DCI)-inactivated proteinases under native conditions (Enghild, J. J., Valnickova, Z., Thøgersen I., and Pizzo, S. V. (1994) J. Biol. Chem. 269, 20159-20166). This study demonstrates that serpin-DCI/proteinase complexes resist dissociation when analyzed in reduced SDS-polyacrylamide gel electrophoresis. Previously, SDS-stable serpin-proteinase complexes have been observed only between serpins and catalytically active proteinases. The stability of these complexes is believed to result from an acyl-ester bond between the active site Ser195 of the proteinase and the alpha-carbonyl group of the scissile bond in the reactive site loop. We have further analyzed the structure of the SDS-stable serpin-proteinase and serpin-DCI/proteinase complexes. The results of these studies demonstrate the presence of the COOH-terminal region of the serpin in both complexes. Since (i) modification of Ser195 does not prevent formation of SDS-stable complexes and (ii) COOH-terminal peptides are present in both complexes, the previously described mechanism does not sufficiently explain the formation of SDS-stable complexes.

Animals↗

PubMed1995

Specific binding of urinary-type plasminogen activator (u-PA) to vitronectin and its role in mediating u-PA-dependent adhesion of U937 cells.

The present paper described interactions of urinary-type plasminogen activator (u-PA) with isolated protein components of the extracellular matrix (ECM) using kinetic and ligand-blotting analyses, as well as adhesion studies with u-PA-saturated U937 monocytic cells. Kinetic analyses showed that fibronectin and laminin were moderately effective at decreasing activation of plasminogen by u-PA (3-4-fold decrease in kcat/Km), while activation was stimulated slightly by collagen types I and IV (2-4-fold increase in kcat/Km). Ligand-blotting experiments using intact immobilized ECM proteins demonstrated that u-PA binds predominantly to vitronectin. This was supported by ELISA studies, which showed concentration dependent, saturable, reversible binding of u-PA to vitronectin (Kd,app. of 97 nM). Limited proteolysis of vitronectin followed by ligand-blotting analysis demonstrated u-PA binding to a specific vitronectin fragment (M(r) 49,000), and binding was shown to occur through the N-terminal fragment of u-PA. N-terminal sequence analysis indicated that this binding fragment of vitronectin originates with Thr-122 and comprises the hemopexin domain, including the heparin-binding region of the vitronectin molecule. Plasminogen activator inhibitor type I did not compete with u-PA for binding to vitronectin, suggesting both molecules may co-localize on vitronectin. In contrast, binding of u-PA to vitronectin was significantly inhibited by plasminogen, suggesting these molecules share a common binding site on vitronectin. In addition to in vitro studies, experiments were performed to assess the contribution of direct binding of u-PA to vitronectin on the adhesive behaviour of U937 cells. Binding of u-PA-saturated U937 cells to vitronectin was inhibited 66% by excess vitronectin, suggesting that direct binding of u-PA to vitronectin is the mechanism by which u-PA-dependent adhesion of U937 cells to vitronectin is mediated.

Amino Acid Sequence↗

PubMed1995

Formation of the alpha 1-microglobulin chromophore in mammalian and insect cells: a novel post-translational mechanism?

alpha 1-Microglobulin is an immunosuppressive plasma protein synthesized by the liver. The isolated protein is yellow-brown, but the hypothetical chromophore has not yet been identified. In this work, it is shown that a human liver cell line, HepG2, grown in a completely synthetic and serum-free medium, secretes alpha 1-microglobulin which is also yellow-brown, suggesting a de novo synthesis of the chromophore by the cells. alpha 1-Microglobulin isolated from the culture medium of insect cells transfected with the gene for rat alpha 1-microglobulin is also yellow-brown, suggesting that the gene carries information about the chromophore. Reduction and alkylation or removal of N- or O-linked carbohydrates by glycosidase treatment did not reduce the colour intensity of the protein. An internal dodecapeptide (amino acid positions 70-81 in human alpha 1-microglobulin) was also yellow-brown. The latter results indicate that the chromophore is linked to the polypeptide. In conclusion, the results suggest that the alpha 1-microglobulin gene carries information activating a post-translational protein modification mechanism which is present in mammalian and insect cells.

Alkylation↗