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Title: Purification and characterization of the glycoprotein hormone. cap alpha. -subunit-like material secreted by HeLa cells

Abstract

The protein secreted by HeLa cells that cross-reacts with antiserum developed against the ..cap alpha..-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 10/sup 5/ ng of ..cap alpha../mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-..cap alpha.. had a composition very similar to that of the urinary hCG ..cap alpha..-subunit. However, comparison of hCG-..cap alpha.. and HeLa-..cap alpha.. demonstrated that the tumor-associated subunit was not identical with its normal counterpart. The purified tumor protein had an apparent molecular weight greater than that of the urinary ..cap alpha..-subunit when analyzed by SDS-PAGE, and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI. Immunoprecipitation and electrophoresis of ..cap alpha..-subunit from HeLa cultures labeled with (/sup 3/H)fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-..cap alpha.. hydrosylates by HPLC confirmed previous reportsmore » that the placental subunit does not contain fucose. The results indicate that, regardless of whether or not a single ..cap alpha..-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors.« less

Authors:
;
Publication Date:
Research Org.:
Univ. of Nebraska Medical Center, Omaha (USA)
OSTI Identifier:
6469000
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (United States); Journal Volume: 27:17
Country of Publication:
United States
Language:
English
Subject:
62 RADIOLOGY AND NUCLEAR MEDICINE; 59 BASIC BIOLOGICAL SCIENCES; GLUCOPROTEINS; AMINO ACID SEQUENCE; HORMONES; RADIOIMMUNOASSAY; BIOCHEMISTRY; CYSTEINE; ELECTROPHORESIS; HCG; HELA CELLS; IODINE 125; METHIONINE; NEOPLASMS; PROTEINS; PURIFICATION; TRITIUM COMPOUNDS; TYROSINE; AMINO ACIDS; BETA DECAY RADIOISOTOPES; CARBOHYDRATES; CARBOXYLIC ACIDS; CHEMISTRY; DAYS LIVING RADIOISOTOPES; DISEASES; DRUGS; ELECTRON CAPTURE RADIOISOTOPES; GLYCOPROTEINS; GONADOTROPINS; HYDROXY ACIDS; IMMUNOASSAY; IMMUNOLOGY; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; LABELLED COMPOUNDS; LIPOTROPIC FACTORS; MOLECULAR STRUCTURE; NUCLEI; ODD-EVEN NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC SULFUR COMPOUNDS; PEPTIDE HORMONES; PITUITARY HORMONES; RADIOASSAY; RADIOIMMUNOLOGY; RADIOISOTOPES; SACCHARIDES; THIOLS; TRACER TECHNIQUES; 550601* - Medicine- Unsealed Radionuclides in Diagnostics; 550201 - Biochemistry- Tracer Techniques

Citation Formats

Cox, G.S., and Rimerman, R.A. Purification and characterization of the glycoprotein hormone. cap alpha. -subunit-like material secreted by HeLa cells. United States: N. p., 1988. Web.
Cox, G.S., & Rimerman, R.A. Purification and characterization of the glycoprotein hormone. cap alpha. -subunit-like material secreted by HeLa cells. United States.
Cox, G.S., and Rimerman, R.A. Tue . "Purification and characterization of the glycoprotein hormone. cap alpha. -subunit-like material secreted by HeLa cells". United States.
@article{osti_6469000,
title = {Purification and characterization of the glycoprotein hormone. cap alpha. -subunit-like material secreted by HeLa cells},
author = {Cox, G.S. and Rimerman, R.A.},
abstractNote = {The protein secreted by HeLa cells that cross-reacts with antiserum developed against the ..cap alpha..-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 10/sup 5/ ng of ..cap alpha../mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-..cap alpha.. had a composition very similar to that of the urinary hCG ..cap alpha..-subunit. However, comparison of hCG-..cap alpha.. and HeLa-..cap alpha.. demonstrated that the tumor-associated subunit was not identical with its normal counterpart. The purified tumor protein had an apparent molecular weight greater than that of the urinary ..cap alpha..-subunit when analyzed by SDS-PAGE, and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI. Immunoprecipitation and electrophoresis of ..cap alpha..-subunit from HeLa cultures labeled with (/sup 3/H)fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-..cap alpha.. hydrosylates by HPLC confirmed previous reports that the placental subunit does not contain fucose. The results indicate that, regardless of whether or not a single ..cap alpha..-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors.},
doi = {},
journal = {Biochemistry; (United States)},
number = ,
volume = 27:17,
place = {United States},
year = {Tue Aug 23 00:00:00 EDT 1988},
month = {Tue Aug 23 00:00:00 EDT 1988}
}