Purification and characterization of the glycoprotein hormone. cap alpha. -subunit-like material secreted by HeLa cells
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.
- Research Organization:
- Univ. of Nebraska Medical Center, Omaha (USA)
- OSTI ID:
- 6469000
- Journal Information:
- Biochemistry; (United States), Vol. 27:17
- Country of Publication:
- United States
- Language:
- English
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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