Purification and partial characterization of a mitogenic protein released from preadipocytes of massively obese subjects

1990 ◽  
Vol 68 (4) ◽  
pp. 764-768 ◽  
Author(s):  
Daniel A. K. Roncari ◽  
Coral E. Thompson
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Radioreceptor Assay ◽  
Massive Obesity ◽  
Epidermal Growth ◽  
Obese Subjects ◽  
High Degree

A protein released into the culture medium by omental preadipocytes of massively obese persons, which stimulates the replication of rat perirenal preadipocytes, has been purified to a high degree. By gel filtration chromatography, the molecular mass of the mitogenic protein was ~66 000 daltons (Da), while on sodium dodecyl sulfate – polyacrylamide gel electrophoresis, two subunits were obtained, relative masses (Mr) of ~31 000 and ~35 000. The isoelectric point of the ~66 000 Da entity was 5.6 ± 0.2. By specific radioreceptor assay, the purified protein was related to epidermal growth factor and transforming growth factor alpha. It was not related to insulin-like growth factors I and II by radioimmunoassay and radioreceptor assay. We propose that the ~66 000 Mr protein, and other mitogenic proteins released by preadipocytes from massively obese persons, act through paracrine–autocrine mechanisms and may play a role in the development of the hyperplasia of enlarged fat cells characteristic of massive corpulence.Key words: preadipocyte, massive obesity, mitogenic, paracrine.

Eicosanoids enhance epidermal growth factor receptor activation and proliferation in glomerular epithelial cells

1992 ◽  
Vol 262 (4) ◽  
pp. F639-F646 ◽  
Author(s):  
A. V. Cybulsky ◽  
P. R. Goodyer ◽  
M. D. Cyr ◽  
A. J. McTavish
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Epithelial Cells ◽  
Egf Receptor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Receptor Activation ◽  
Scatchard Analysis ◽  
Glomerular Epithelial Cells ◽  
Epidermal Growth

Proliferation of glomerular epithelial cells (GEC) and release of prostaglandins (PG) and thromboxane (Tx) A2 may occur in glomerular injury. We studied the relationship of eicosanoids to epidermal growth factor (EGF)-induced proliferation of rat GEC in culture. After 48 h of serum-deprivation, EGF stimulated [3H]thymidine incorporation ninefold above serum-deprived cells. Inhibition of cyclooxygenase with indomethacin or of Txsynthase with OKY-046 decreased the proliferative effect of EGF by 50 and 38%, respectively. The effect of indomethacin was reversed by addition of PGE2. Synthesis of PGE2, PGF2 alpha, and TxA2 by serum-deprived GEC was not enhanced by EGF. Scatchard analysis of 125I-EGF binding to GEC demonstrated two populations of EGF receptors; the high-affinity site had a dissociation constant (Kd) of 444 pM and 24,864 receptors/cell. EGF receptor autophosphorylation (reflecting receptor activation) was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of GEC membrane proteins with anti-phosphotyrosine antibody. EGF increased phosphorylation of a protein of approximately 170 kDa, which comigrated with proteins immunoprecipitated from [35S]methionine-labeled GEC with antibodies to EGF receptor. Indomethacin and OKY-046 decreased the EGF-dependent phosphorylation of the 170-kDa protein, and this decrease was overcome by addition of PGE2. Indomethacin and OKY-046 did not, however, reduce 125I-EGF binding. Thus, in GEC, the basal synthesis of eicosanoids enhanced EGF-induced proliferation. This effect appears to be due to enhancement of EGF receptor activation.

scholarly journals Isolation and partial characterization of canine epidermal growth factor/urogastrone

1985 ◽  
Vol 229 (3) ◽  
pp. 611-619 ◽  
Author(s):  
R Kobayashi ◽  
J R Reeve ◽  
J H Walsh
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Mouse Fibroblast ◽  
A431 Cells ◽  
Peptide Component ◽  
Epidermal Growth

Canine epidermal growth factor (EGF)/urogastrone was partially purified from dog urine by fractional precipitation with (NH4)2SO4, ion-exchange chromatography with DEAE-cellulose DE-52, gel filtration with Sephadex G-50, and a second DE-52 chromatography, to yield receptor-competing activity equivalent to 13 micrograms of standard mouse EGF/litre of starting urine. The purification was monitored by a competitive radioreceptor assay using fixed monolayers of A431 cells. The partially purified canine EGF/urogastrone demonstrated a growth-stimulating activity in 3T3 mouse fibroblast cells as potent as mouse EGF. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one major peptide component with an Mr similar to that of mouse EGF, and two minor peptides of slightly higher Mr. The major peptide component was isolated after reduction and its amino acid composition was determined.

scholarly journals COMPARISON OF EXTRACELLULAR SECRETION OF RECOMBINANT HUMAN EPIDERMAL GROWTH FACTOR USING TORA AND PELB SIGNAL PEPTIDES IN ESCHERICHIA COLI BL21 (DE3)

2019 ◽  
pp. 81-84 ◽  
Author(s):  
RIMA MELATI ◽  
ANNISA INDRIYANI ◽  
SHABARNI GAFFAR ◽  
SRIWIDODO ◽  
IMAN PERMANA MAKSUM
Keyword(s):  
Escherichia Coli ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Signal Peptides ◽  
Extracellular Expression ◽  
E Coli ◽  
Human Epidermal Growth Factor ◽  
Epidermal Growth

Objective: The objective of this study was to evaluate two signal peptides (TorA and PelB), representing the most common secretion pathways in Escherichia coli, for their ability to secrete recombinant human epidermal growth factor (rhEGF) protein in the extracellular expression. Methods: E. coli BL21 (DE3) as the host cell to be transformed using recombinant plasmid pD881-TorA the consensus already containing hEGF gene and the signal peptide TorA or PelB, then expressed by L-rhamnose induction. rhEGF purified by heat treatment and ion-exchange chromatography. The hEGF protein was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ELISA. Results: The result showed that PelB was secreting more hEGF protein compared to TorA with protein expression results of 48.2 μg/L and purification results of 0.360 μg/L, with a purity level of 83%. Conclusion: The results of this study explain in extracellular expression of hEGF protein in E. coli, PelB helps hEGF protein secretion to culture media better than TorA.

scholarly journals Purification, Characterization, and Sequence Analysis of 2-Aminomuconic 6-Semialdehyde Dehydrogenase from Pseudomonas pseudoalcaligenes JS45

1998 ◽  
Vol 180 (17) ◽  
pp. 4591-4595 ◽  
Author(s):  
Zhongqi He ◽  
John K. Davis ◽  
Jim C. Spain
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Gene Encoding ◽  
Multiple Sequence ◽  
Pseudomonas Pseudoalcaligenes ◽  
Semialdehyde Dehydrogenase ◽  
Cell Extracts ◽  
High Degree

ABSTRACT 2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by Pseudomonas pseudoalcaligenes JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD+. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases.

scholarly journals Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells.

1983 ◽  
Vol 3 (8) ◽  
pp. 1343-1352 ◽  
Author(s):  
A R Frackelton ◽  
A H Ross ◽  
H N Eisen
Keyword(s):  
Monoclonal Antibodies ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Leukemia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hydroxyl Group ◽  
Atp Citrate Lyase ◽  
Ionic Detergents ◽  
Epidermal Growth

Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.

scholarly journals Further characterization of galactosylhydroxylysyl glucosyltransferase from chick embryos. Amino acid composition and acceptor specificity

1978 ◽  
Vol 175 (2) ◽  
pp. 737-742 ◽  
Author(s):  
H Anttinen ◽  
R Myllylä ◽  
K I Kivirikko
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Structural Similarity ◽  
Enzyme Preparations ◽  
Glucose Transfer ◽  
Acceptor Specificity ◽  
High Degree ◽  
Glucose Derivative

A modified purification procedure, consisting of affinity chromatographies on concanavalin A-agarose, collagen-agarose and UDP-glucose-derivative-agarose and one gel filtration, is reported for galactosylhydroxylysyl glucosyltransferase. The enzyme obtained is entirely pure when studied by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme protein was rich in glutamic acid + glutamine, aspartic acid + asparagine, glycine and alanine. The enzyme catalysed no significant glucose transfer to any of the glycoproteins tested, except for collagens. This included all the glycoproteins that have previously served as glucosyl acceptors for impure enzyme preparations, thus indicating a high degree of specificity of the enzyme for galactosylhydroxylysine. Galactosylsphingosine would act as a glucosyl acceptor, however. This compound has a close structural similarity to galactosylhydroxylysine in that they both have an unsubstituted amino group next to the hydroxy group to which the galactose is attached.

A sensitive enzyme immunoassay system of rat epidermal growth factor in biological fluids and tissue extracts

1989 ◽  
Vol 120 (5) ◽  
pp. 616-623 ◽  
Author(s):  
Takashi Joh ◽  
Makoto Itoh ◽  
Naoji Yasue ◽  
Tadahisa Miyamoto ◽  
Akira Iwai ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Enzyme Immunoassay ◽  
Biological Fluids ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Sensitivity ◽  
Soft Agar ◽  
Epidermal Growth ◽  
Sandwich Enzyme Immunoassay

Abstract. Rat epidermal growth factor was purified from rat submandibular glands to obtain specific antiserum for the establishment of an immunoassay system. Purified rat epidermal growth factor showed a single peak on reverse phase HPLC and a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis at mol wt 5100 in the presence of 2-mercaptoethanol and at mol wt 41000 in the absence of 2-mercaptoethanol. Antibody against rat epidermal growth factor showed crossreactivities with mouse and human epidermal growth factors on soft agar-double immunodiffusion test. The established sandwich enzyme immunoassay for rat epidermal growth factor had a high sensitivity (500 fg/tube), which made it possible to measure minute amounts of endogenous rat epidermal growth factor without pretreatment. Physiological concentrations of rat epidermal growth factor in rat biological fluids and tissues were determined. Species differences in physiological distributions of epidermal growth factor are discussed.

Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells

1983 ◽  
Vol 3 (8) ◽  
pp. 1343-1352
Author(s):  
A R Frackelton ◽  
A H Ross ◽  
H N Eisen
Keyword(s):  
Monoclonal Antibodies ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Leukemia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hydroxyl Group ◽  
Atp Citrate Lyase ◽  
Ionic Detergents ◽  
Epidermal Growth

Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.

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