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M X Sliwkowski

Publications and source records attributed to M X Sliwkowski.

At least 55 records · Page 3Linked to original sources
PubMed1994

Coexpression of erbB2 and erbB3 proteins reconstitutes a high affinity receptor for heregulin.

The heregulin/neu differentiation factor gene products were purified and cloned based on their ability to stimulate the phosphorylation of a 185-kDa protein in human breast carcinoma cell lines known to express erbB2. However, not all cells that express erbB2 respond to heregulin, indicating that other components besides erbB2 may be required for heregulin binding. Cells that are transfected with the closely related receptor, erbB3, display a single class of lower affinity heregulin binding sites than has been previously observed on breast carcinoma cell lines. Little or no stimulation of tyrosine phosphorylation in response to heregulin occurs in cells that are transfected with erbB3 alone. Transfection of cells with erbB3 and erbB2 reconstitutes a higher affinity binding receptor, which is also capable of generating a tyrosine phosphorylation signal in response to heregulin. A monoclonal antibody to erbB2 will inhibit heregulin activation of tyrosine phosphorylation and binding in cells transfected with both receptors but not with erbB3 alone. In cells expressing erbB2 and erbB3, both proteins become tyrosine-phosphorylated upon interaction with heregulin. Direct interaction between heregulin and the two proteins was demonstrated by chemical cross-linking experiments using 125I-heregulin followed by immunoprecipitation with antibodies specific for erbB2 or erbB3.

Animals↗
PubMed1994

The erbB3 gene product is a receptor for heregulin.

ErbB3 is a member of the epidermal growth factor (EGF) receptor subfamily of receptor tyrosine kinases and is believed to be a receptor for an unknown ligand. We have tested the possibility that heregulin, a growth factor possessing an EGF-like domain, is a ligand for ErbB3. We have found that the iodinated recombinant EGF-like domain of heregulin-beta 1 (125I-rHRG beta 1(177-244) bound specifically to insect cell-expressed bovine ErbB3 with a dissociation constant of 0.85 nM. Moreover, 125I-rHRG beta 1(177-244) bound to NIH3T3 fibroblasts stably transfected with bovine erbB3 with a dissociation constant of 60 pM, but did not bind to parental cells. 125I-rHRG beta 1(177-244) could be chemically cross-linked to a 170-180 kDa protein in erbB3-transfected fibroblasts, and the cross-linked product could be immunoprecipitated with antibodies specific for ErbB3. Finally, rHRG beta 1 stimulated the tyrosine phosphorylation of both ErbB3 and endogenous p185erbB2/neu in transfectants but not in parental cells. We conclude that ErbB3 is a receptor for HRG and is capable of mediating HRG-stimulated tyrosine phosphorylation of itself and p185erbB2/neu in cells that express both receptors.

3T3 Cells↗
PubMed1994

Genetically engineered proinsulin constitutively processed and secreted as mature, active insulin.

The conversion of human proinsulin to insulin occurs only in specialized cells which contain the appropriate processing enzymes. To allow proinsulin processing to occur in a wide variety of cell types, we engineered human proinsulin to be cleaved in the constitutive secretory pathway. Using site-directed mutagenesis, we have introduced furin consensus cleavage sequences (Arg-X-Lys-Arg) into the human proinsulin cDNA. These mutations allowed for efficient proteolytic maturation of human proinsulin to insulin within cells containing only a constitutive pathway of secretion. Additionally, a naturally occurring mutation (histidine B10 to aspartic acid) yields a form of human insulin which accumulates 10- to over 100-fold more mature insulin when compared to the mutants lacking this change. Engineering furin-specific cleavage sites into each junction of the human proinsulin cDNA results in the secretion of peptides that display the expected molecular weights for the A and B chains of insulin. The accumulation of mature, processed, human insulin was measured in the supernatants by radioimmunoassay, and the bioactivity of this molecule was measured by its ability to stimulate autophosphorylation of the insulin receptor. Our results suggest that any cell type might be engineered to produce mature, active, and stable insulin in the constitutive pathway of secretion.

3T3 Cells↗
PubMed1992

Identification of heregulin, a specific activator of p185erbB2.

The proto-oncogene designated erbB2 or HER2 encodes a 185-kilodalton transmembrane tyrosine kinase (p185erbB2), whose overexpression has been correlated with a poor prognosis in several human malignancies. A 45-kilodalton protein heregulin-alpha (HRG-alpha) that specifically induced phosphorylation of p185erbB2 was purified from the conditioned medium of a human breast tumor cell line. Several complementary DNA clones encoding related HRGs were identified, all of which are similar to proteins in the epidermal growth factor family. Scatchard analysis of the binding of recombinant HRG to a breast tumor cell line expressing p185erbB2 showed a single high affinity binding site [dissociation constant (Kd) = 105 +/- 15 picomolar]. Heregulin transcripts were identified in several normal tissues and cancer cell lines. The HRGs may represent the natural ligands for p185erbB2.

Amino Acid Sequence↗
PubMed1990

Serological evaluation of Escherichia coli-expressed human T-cell leukemia virus type I env, gag p24, and tax proteins.

Three proteins (env, gag, and tax) encoded by the human T-cell leukemia virus type I (HTLV-I) genome were cloned and expressed in Escherichia coli. The env protein contained a substantial part of the gp46 domain and a majority of the p21e domain. The gag protein contained all of p24 and portions of p19 and p15. In addition to these two structural proteins, a full-length tax (p40X) construct was obtained. All three recombinant proteins were purified to near homogeneity. When used in an immunoblot assay, the three recombinant proteins detected antibodies in more HTLV-I antibody-positive patient sera than did the corresponding native proteins. Antibodies to at least two of these three different gene products were detected in 98.4% of adult T-cell leukemia patients, 100% of HTLV-I-associated myelopathy patients, 97.4% of asymptomatic carriers, and 94% of uncharacterized HTLV-I-positive patients. Antibody to recombinant tax was found in 4.9% of adult T-cell leukemia patients, whereas antibody to recombinant env could not be detected. These recombinant proteins from three different gene products may be useful in detecting or confirming the presence of antibodies to HTLV-I.

Adult↗
PubMed1988

Selenoprotein A of the clostridial glycine reductase complex: purification and amino acid sequence of the selenocysteine-containing peptide.

A selenium-containing protein, selenoprotein A, is an essential component of the clostridial glycine reductase complex. This enzyme complex catalyzes the reductive deamination of glycine, which is coupled to the esterification of orthophosphate resulting in the formation of ATP. Sequence information was obtained by automated Edman degradation of peptides generated by digesting carboxamidomethylated selenoprotein A with chymotrypsin or trypsin or with endoproteinase Arg-C followed by Staphylococcus aureus V8 protease. The sequence near the selenocysteine (Sec) residue is -Cys-Phe-Val-Sec-Thr-Ala-Ala-Gly-Ala-Met-Asp-Leu-Glu-Asn-Glu-Lys-. Selenium-containing peptides isolated from digests of carboxamidomethylated selenoprotein A with trypsin or endoproteinase Arg-C were found to be blocked at the amino terminus. The sequence of the selenocysteine-containing peptide from selenoprotein A shows no homology with those of two other selenoproteins, glutathione peroxidase and formate dehydrogenase.

Amino Acid Oxidoreductases↗
PubMed1988

Selenium-dependent glycine reductase: differences in physicochemical properties and biological activities of selenoprotein A components isolated from Clostridium sticklandii and Clostridium purinolyticum.

The selenoprotein A component of the glycine reductase complex of Clostridium sticklandii was shown to differ in certain properties from the selenoprotein A produced by a purine-fermenting organism, Clostridium purinolyticum. Both proteins contain one selenocysteine and two cysteine residues.

Amino Acid Oxidoreductases↗
PubMed1987

Purification and immunological studies of selenoprotein A of the clostridial glycine reductase complex.

One of the essential catalytic components of the clostridial glycine reductase complex is a selenium-containing protein, selenoprotein A. An improved method for the purification of selenoprotein A has been developed that yields milligram quantities of the protein in a few relatively simple steps. Ferredoxin and rubredoxin can be recovered in pure form as by-products of the procedure. The high resolving capabilities of an anion exchange high pressure liquid chromatography step were exploited in these purification protocols. For effective antibody production, the antigenicity of selenoprotein A was increased by coupling the pure protein via its sulfhydryl/selenol group(s) to the amino groups of bovine serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester. The high titer sheep antisera that were elicited were used to study the mechanisms of selenium incorporation into selenoprotein A. Immunoblot analysis of sodium dodecyl sulfate-polyacrylamide gels was employed to monitor the synthesis of selenoprotein A by Clostridium sticklandii as a function of growth conditions. Cells grown under limiting conditions (1 nM) of selenium contained only 1-2% of normal levels of active selenoprotein A and no precursor forms were detected after DEAE-high pressure liquid chromatography fractionation of extracts. Conversely, cells grown with optimal (1 microM) selenium levels contained maximal amounts of seleno-protein A and expressed full glycine reductase activity.

Amino Acid Oxidoreductases↗
PubMed1985

Incorporation and distribution of selenium into thiolase from Clostridium kluyveri.

Clostridium kluyveri incorporates selenium as selenomethionine into its acetoacetyl-CoA thiolase when grown in media containing normal sulfur-to-selenium ratios. Antibodies raised against the purified enzyme permitted quantitative immunoprecipitation of thiolase from crude cell extracts and thus facilitated the systematic analysis of the effects of wide variation in sulfur-to-selenium ratios on selenium incorporation into the enzyme. The extent of incorporation of selenium into thiolase was found to be dependent on the form of selenium supplied. When [75Se]selenomethionine was the source of selenium, the incorporation of selenium into thiolase was inversely proportional to the level of added methionine. However, similar levels of methionine failed to decrease the incorporation of selenium from selenite. To study the location of selenomethionine and methionine residues in the polypeptide chain of the enzyme, thiolase was prepared from cells cultured in the presence of H2 35SO4 or Na2 75SeO3. The 35S- or 75Se-labeled protein was treated with trypsin and the resulting peptides were isolated by reverse phase high performance liquid chromatography. The peptide maps of the enzyme indicated that selenium was distributed throughout the primary structure in a manner that paralleled methionine. From these studies, it is concluded that selenium occurs in thiolase adventitiously and is not required for any biological function.

Acetyl-CoA C-Acetyltransferase↗
PubMed1985

Labeling of cysteine-containing peptides with 2-nitro-5-thiobenzoic acid.

A method for specific labeling of cysteine-containing peptides has been developed using Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Prior to cleavage with proteases or chemical reagents, proteins are reacted with DTNB, resulting in the formation of a mixed disulfide between the protein sulfhydryl group and 2-nitro-5-thiobenzoic acid (TNB). The formation of the mixed disulfide introduces a chromophore, with an absorbance peak at 328 nm. By monitoring peptide maps generated by HPLC at 210 and 328 nm, peptides containing cysteine residues are readily identified. The stability of the derivative was tested using glutathione-TNB as a model compound. Glutathione-TNB is stable to conditions used for CNBr cleavage, as well as those for tryptic cleavage. The TNB label may also increase the hydrophobicity of small peptides, which otherwise might not bind to reverse-phase matrices. This was the case for an oxidatively modified tetrapeptide isolated from Escherichia coli glutamine synthetase.

Chromatography, High Pressure Liquid↗
PubMed1984

Kinetic mechanism and specificity of bovine milk sulphydryl oxidase.

Sulphydryl oxidase is known to catalyse the synthesis de novo of disulphide bonds in a variety of thiol-containing compounds. Reduced glutathione is the best thiol substrate; however, D- and L-cysteine, cysteamine and N-acetyl-L-cysteine, as well as cysteine-containing peptides and proteins, are also effectively oxidized. In contrast, oxidation of the thiol groups of mercaptoethanol, mercaptopyridine, dithiothreitol, dithioerythritol, mercaptoacetate, mercaptopropionate or lipoic acid is not detectably catalysed. In bovine milk, sulphydryl oxidase is closely associated with another glutathione-metabolizing enzyme, gamma-glutamyltransferase. Covalent chromatography of crude preparations on cysteinylsuccinamidopropyl-glass resolves the oxidase from the transferase, thus permitting the kinetic characterization of glutathione oxidation. Initial-rate data imply a Ter Bi substituted-enzyme mechanism, and the observed substrate inhibition by thiols suggest that O2 binds first. Independent, non-kinetic, data, namely the immobilization of sulphydryl oxidase on cysteinyl-matrices, support formation of a mixed-disulphide intermediate between the thiol and enzyme, as predicted by the proposed mechanism. The enzyme-catalysed reaction appears not to be mediated via a superoxide intermediate, since O2 consumption is not affected by the presence of Nitro Blue Tetrazolium. FAD, NAD+, NADP+ and Nitro Blue Tetrazolium are all inactive as electron acceptors for sulphydryl oxidase catalysis.

Acetylcysteine↗
PubMed1984

Simultaneous single-step purification of thiolase and NADP-dependent 3-hydroxybutyryl-CoA dehydrogenase from Clostridium kluyveri.

Thiolase and NADP-dependent 3-hydroxybutyryl-CoA dehydrogenase from Clostridium kluyveri were purified by ion-exchange chromatography to near homogeneity in a simultaneous, single-step procedure. The yield of both enzymes was greater than 80%. Thiolase was purified approximately 8-fold with sp act 115 units/mg, whereas 3-hydroxybutyryl-CoA dehydrogenase was purified 14-fold with sp act 292 units/mg. Isoelectric points of the enzymes are 7.7 for thiolase and 7.8 for 3-hydroxybutyryl-CoA dehydrogenase. Milligram quantities of each of these enzymes are readily obtained from this fatty acid-producing organism.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1983

Resolution of sulphydryl oxidase from gamma-glutamyltransferase in bovine milk by covalent chromatography on cysteinylsuccinamidopropyl-glass.

1. Sulphydryl oxidase from bovine milk was purified by covalent affinity chromatography on cysteinylsuccinamidopropyl-glass. Selective immobilization of the oxidase occurs through formation of a mixed disulphide between the enzyme and the substrate cysteinyl-glass matrix. Reductive elution of the bound protein can be accomplished with small thiols such as reduced glutathione (GSH), dithiothreitol or cysteine. This method leads to approx. 4000-fold purification of the enzyme from whey. Furthermore, complete resolution of sulphydryl oxidase from gamma-glutamyltransferase was achieved with this procedure. 2. Antibodies prepared against this purified enzyme quantitatively precipitated 95% of the GSH-oxidative activity from detergent-solubilized skim-milk membranes, whereas 100% of the transferase activity remained in the supernatant fraction; these findings confirmed the distinction between these two enzymes. 3. Reverse-phase high-pressure-liquid-chromatographic analyses of assay mixtures containing both enzymes revealed an array of GSH derivatives generated by a combination of the oxidative and hydrolytic activities. However, purified sulphydryl oxidase yielded only GSSG with concomitant stoichiometric loss of GSH. 4. The chromatographic method described is simple and reproducible, and may be applicable to isolation of sulphydryl oxidase from other tissues.

Animals↗