Structural and functional studies on the sodium- and chloride-coupled gamma-aminobutyric acid transporter: deglycosylation and limited proteolysis.
The sodium- and chloride-coupled gamma-aminobutyric transporter, an 80-kDa glycoprotein, has been subjected to deglycosylation and limited proteolysis. The treatment of the 80-kDa band with endoglycosidase F results in its disappearance and reveals the presence of a polypeptide with an apparent molecular mass of about 60 kDa, which is devoid of 125I-labeled wheat germ agglutinin binding activity but is nevertheless recognized by the antibodies against the 80-kDa band. Upon limited proteolysis with papain or Pronase, the 80-kDa band was degraded to one with an apparent molecular mass of about 60 kDa. This polypeptide still contains the 125I-labeled wheat germ agglutinin binding activity but is not recognized by the antibody. The effect of proteolysis on function was examined. The transporter was purified by use of all steps except that for the lectin chromatography [Radian, R., Bendahan, A., & Kanner, B.I. (1986) J. Biol. Chem. 261, 15437-15441]. After papain treatment and lectin chromatography, gamma-aminobutyric transport activity was eluted with N-acetylglucosamine. The characteristics of transport were the same as those of the pure transporter, but the preparation contained instead of the 80-kDa polypeptide two fragments of about 66 and 60 kDa. The ability of the anti-80-kDa antibody to recognize these fragments was relatively low. The observations indicate that the transporter contains exposed domains which are not important for function.
Kanner
B I
BI
Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
Keynan
S
S
Radian
R
R
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D050485:GABA Plasma Membrane Transport Proteins; D006026:Glycoside Hydrolases; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D027361:Organic Anion Transporters; D010206:Papain; D012964:Sodium / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D002712:Chlorides; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D027361:Organic Anion Transporters; D005680:gamma-Aminobutyric Acid; D012964:Sodium; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D010206:Papain