Preparation and application of a pentamannosyl monophosphate-bovine serum albumin conjugate.
Pentamannosyl monophosphate, derived from Hansenula holstii O-phosphomannan, was conjugated to bovine serum albumin by reductive amination. The conjugate inhibited the binding of the porcine testis mannose 6-phosphate receptor to the insoluble phosphomannan core. A mannose 6-phosphate receptor with a molecular weight of 200,000 was purified from porcine liver membranes, using an affinity matrix of the conjugate attached to Sepharose 4B. Rabbits were immunised with the conjugate, and the antisera were purified on a phosphomannan core-Sepharose 4B column in order to give an antibody which was specific for the 6-phosphate group and the equatorial HO-4 of D-mannose 6-phosphate. On Western blot analysis using the purified antibodies, ovalbumin, which contained a typical high-mannose type of oligosaccharide, was not recognised. However, a testicular glycoprotein fraction formed an immunostaining band. These results indicate the effectiveness of the conjugate as a ligand for mannose 6-phosphate receptors. The antibodies highly specific for mannose 6-phosphate may be used to detect or purify lysosomal enzymes.
Baba
T
T
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
Watanabe
K
K
Yonezawa
N
N
Hiroto
M
M
Arai
Y
Y
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006600:Hexosephosphates / Q000138:chemical synthesis*; D008099:Liver / Q000378:metabolism; D008297:Male; D008360:Mannosephosphates / Q000138:chemical synthesis* / Q000276:immunology / Q000378:metabolism; D008970:Molecular Weight; D017527:Receptor, IGF Type 2; D012710:Serum Albumin, Bovine / Q000138:chemical synthesis*; D013552:Swine; D013737:Testis / Q000378:metabolism
D000906:Antibodies; D002352:Carrier Proteins; D006600:Hexosephosphates; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; C054881:pentamannosyl phosphate substituted bovine serum albumin; D012710:Serum Albumin, Bovine; C027693:mannose-6-phosphate