Metabolic behavior of cell surface biotinylated proteins.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Hare
J F
JF
Department of Biochemistry, School of Medicine, Oregon Health Sciences University, Portland 97201.
Lee
E
E
D000614:Aminocaproates; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006207:Half-Life; D006834:Hydrazines; D007525:Isoelectric Focusing; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D013388:Succinimides; D013462:Sulfur Radioisotopes; D014316:Tritium
D000614:Aminocaproates; D006834:Hydrazines; D008565:Membrane Proteins; D013388:Succinimides; D013462:Sulfur Radioisotopes; D014316:Tritium; C059612:N-biotinoyl-N'-(maleimidohexanoyl)hydrazine; D001710:Biotin; C048592:biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester; D008715:Methionine