ABSTRACT
The purple acid phosphatase (PAP) from bovine spleen has been shown to exist as a single ca. 36-kDa polypeptide in intact spleen tissue. The previously isolated microheterogeneous complex of 15-kDa and 23- or 21-kDa subunits appears to arise from proteolytic cleavage of an exposed, highly variable loop in the polypeptide chain. Small amounts of a single polypeptide form, presumed to be the native form of the enzyme, have been obtained; this has permitted its optical and EPR spectra and fundamental kinetic properties to be determined. The most notable difference between the native and two-subunit forms of PAP is a ca. 3-fold higher enzymatic activity for the latter, which is due to a simple increase in Vmax. The two forms are very similar spectroscopically and chemically and appear to differ only in the loss of a highly antigenic ca. five amino acid segment of the polypeptide between positions 155 and 160 but not in NH2-terminal sequence or in carbohydrate content. Analysis of published sequence data suggests that the existence of an exposed highly antigenic loop at positions corresponding to 155-161 of the spleen PAP sequence is a relatively general feature of PAP's. Trypsin and chymotrypsin cleave both bovine spleen PAP and uteroferrin, apparently in this region, with significant enhancement of enzymatic activity.
Subject(s)
Acid Phosphatase/isolation & purification , Spleen/enzymology , Acid Phosphatase/chemistry , Acid Phosphatase/metabolism , Amino Acid Sequence , Amino Acids/analysis , Animals , Blotting, Western , Carbohydrates/analysis , Cattle , Circular Dichroism , Electron Spin Resonance Spectroscopy , Endopeptidases/pharmacology , Isoenzymes , Kinetics , Metalloproteins/metabolism , Molecular Sequence Data , Molybdenum/pharmacology , Phosphates/pharmacology , Protease Inhibitors/pharmacology , Sequence Analysis , Spectrophotometry , Tartrate-Resistant Acid PhosphataseABSTRACT
The primary structures of purple acid phosphatase and uteroferrin, two iron-binding glycoproteins isolated from beef spleen and porcine uterine fluids, respectively, have been examined by a combination of tandem mass spectrometry and classical Edman sequencing methods. Reported here are amino acid sequence data covering more than 90% of the primary structures for these two proteins. The sequence data reveal an unexpectedly high degree of homology, greater than 90%, for these two proteins.
