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Physical and Chemical Characterization of Pig Kidney Particulate Aminopeptidase
Author(s) -
Wacker Hans,
Lehky Pavel,
Fischer Edmond H.,
Stein Eric A.
Publication year - 1971
Publication title -
helvetica chimica acta
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.74
H-Index - 82
eISSN - 1522-2675
pISSN - 0018-019X
DOI - 10.1002/hlca.19710540206
Subject(s) - chemistry , chromatography , sialic acid , fucose , aminopeptidase , hydrolysis , partial specific volume , molecular mass , enzyme , biochemistry , leucine , galactose , amino acid
Pig kidney particulate aminopeptidase (EC 3.4.1.2) was purified by a modification of the procedure of Wachsmuth et al. [3] to a state of homogeneity according to criteria of ultracentrifugation and polyacrylamide gel electrophoresis, and some of its physical and chemical properties were determined. The purified enzyme (specific activity 30 ± 3 μmoles leucine‐ p ‐nitroanilide hydrolyzed/min/mg) has a s 20,w = 9.82 S at pH 8 and a molecular weight of about 280,000 as determined by high speed sedimentation equilibrium. Divergence between the number‐, weight‐ and z‐ average molecular weights, and a sharp decrease of these values at low protein concentration, suggest that one is dealing with an associating‐dissociating system. Amino acid analysis revealed the presence of considerable quantities of carbohydrates in the enzyme. Colorimetric, gas chromatographic and enzymatic analyses demonstrated the presence of glucosamine, galactose, mannose, fucose and sialic acid residues in the ratio of 1:0.89:0.75: 0.13: 0.13, amounting to ca . 400 residues or 20% (56,000 daltons) of the molecular weight. With the exception of sialic acid, the carbohydrate content was remarkably constant from preparation to preparation. Analyses by both atomic absorption spectrometry and the dithizone method showed that zinc–the only metal found in significant amount–was always present in the ratio of 2 atoms per molecule. It is therefore proposed that pig kidney particulate aminopeptidase is a Zn‐containing glycoportein.