Metabolic behavior of cell surface biotinylated proteins

Journal Article · Tue Jan 24 04:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00428a024· OSTI ID:5563436

Hare, J F; Lee, E ^[1]

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1. Oregon Health Sciences Univ., Portland (USA)

The turnover of proteins on the surface of cultured mammalian cells was measured by a new approach. Reactive free amino or sulfhydryl groups on surface-accessible proteins were derivatized with biotinyl reagents and the proteins solubilized from culture dishes with detergent. Solubilized, biotinylated proteins were then adsorbed onto streptavidin-agarose, released with sodium dodecyl sulfate and mercaptoethanol, and separated on polyacrylamide gels. Biotin-epsilon-aminocaproic acid N-hydroxysuccinimide ester (BNHS) or N-biotinoyl-N'-(maleimidohexanoyl)hydrazine (BM) were the derivatizing agents. Only 10-12 bands were adsorbed onto streptavidin-agarose from undervatized cells or from derivatized cells treated with free avidin at 4 degrees C. Two-dimensional isoelectric focusing-sodium dodecyl sulfate gel electrophoresis resolved greater than 100 BNHS-derivatized proteins and greater than 40 BM-derivatized proteins. There appeared to be little overlap between the two groups of derivatized proteins. Short-term pulse-chase studies showed an accumulation of label into both groups of biotinylated proteins up until 1-2 h of chase and a rapid decrease over the next 1-5 h. Delayed appearance of labeled protein at the cell surface was attributed to transit time from site of synthesis. The unexpected and unexplained rapid disappearance of pulse-labeled proteins from the cell surface was invariant for all two-dimensionally resolved proteins and was sensitive to temperature reduction to 18 degrees C. Long-term pulse-chase experiments beginning 4-8 h after the initiation of chase showed the disappearance of derivatized proteins to be a simple first-order process having a half-life of 115 h in the case of BNHS-derivatized proteins and 30 h in the case of BM-derivatized proteins.

• OSTI ID: 5563436
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:2; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550501* -- Metabolism-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
AZOLES
BIOLOGICAL HALF-LIFE
BIOTIN
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
DRUGS
ELECTROPHORESIS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYDRAZINE
HYDROGEN COMPOUNDS
IMIDAZOLES
ISOTOPE APPLICATIONS
ISOTOPES
LIPOTROPIC FACTORS
MEMBRANE PROTEINS
MEMBRANES
METABOLISM
METHIONINE
MOLECULAR WEIGHT
NITROGEN COMPOUNDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PROTEINS
SULFUR ISOTOPES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VITAMIN B GROUP
VITAMINS