title: Isolation by zinc-affinity chromatography of the histidine-proline-rich-glycoprotein molecule associated with rabbit skeletal muscle AMP deaminase. Evidence that the formation of a protein-protein complex between the catalytic subunit and the novel component is critical for the stability of the enzyme.
abstract: The histidine-proline-rich glycoprotein (HPRG) component of rabbit skeletal muscle AMP deaminase under denaturing and reducing conditions specifically binds to a Zn(2+)-charged affinity column and is only eluted with an EDTA-containing buffer that strips Zn(2+) from the gel. The isolated protein is homogeneous showing an apparent molecular weight (MW) of 95000 and the N-terminal sequence L-T-P-T-D-X-K-T-T-K-P-L-A-E-K-A-L-D-L-I, corresponding to that of rabbit plasma HPRG. The incubation with peptide-N-glycosidase F promotes the reduction of the apparent MW of isolated HPRG to 70000, characterizing it as a N-glycosylated protein. The separation from AMP deaminase of an 85-kDa component with a blocked N terminus is observed when the enzyme is applied to the Zn-charged column under nondenaturing conditions. On storage under reducing conditions, this component undergoes an 85- to 95-kDa transition yielding a L-T-P-T-D-X-K-T-T-K-P-L N-terminal sequence, suggesting that the shift in the migration on SDS/PAGE as well as the truncation of the protein at its N terminus are promoted by the reduction of a disulfide bond present in freshly isolated HPRG. The separation of HPRG induces a marked reduction in the solubility of AMP deaminase, strongly suggesting a role of HPRG in assuring the molecular integrity of the enzyme.
authors.affiliation: Dipartimento di Scienze dell'Uomo e dell'Ambiente, Chimica e Biochimica Medica, Università di Pisa, via Roma 55, 56126, Pisa, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Ranieri-Raggi
authors.forename: Daniela
authors.initials: D
authors.lastname: Martini
authors.forename: Antonietta R M
authors.initials: AR
authors.lastname: Sabbatini
authors.forename: Arthur J G
authors.initials: AJ
authors.lastname: Moir
authors.forename: Antonio
authors.initials: A
authors.lastname: Raggi
mesh_terms: D000659:AMP Deaminase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006023:Glycoproteins / Q000302:isolation & purification*; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism*; D009569:Nitric Oxide / Q000737:chemistry; D009589:Nitrogen Oxides; D011392:Proline / Q000031:analogs & derivatives* / Q000737:chemistry; D020836:Protein Structure, Quaternary; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D011817:Rabbits; D015032:Zinc; D000099053:Histidine-Rich Glycoprotein
chemical_list: D006023:Glycoproteins; D009589:Nitrogen Oxides; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein; C102043:proline-nitric oxide; D009569:Nitric Oxide; D006639:Histidine; D011392:Proline; D000659:AMP Deaminase; D015032:Zinc