Purification and characterization of aspartic proteinase from sunflower seeds.
Aspartic proteinases were purified from sunflower seed extracts by affinity chromatography on a pepstatin A-EAH Sepharose column and by Mono Q column chromatography. The final preparation contained three purified fractions. SDS-PAGE showed that one of the fractions consisted of disulfide-bonded subunits (29 and 9 kDa), and the other two fractions contained noncovalently bound subunits (29 and 9 kDa). These purified enzymes showed optimum pH for hemoglobinolytic activity at pH 3.0 and were completely inhibited by pepstatin A like other typical aspartic proteinases. Sunflower enzymes showed more restricted specificity on oxidized insulin B chain and glucagon than other aspartic proteinases. The cDNA coding for an aspartic proteinase was cloned and sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 440 amino acid residues with a molecular mass of 47,559 Da. The difference between the molecular size of purified enzymes and of the mature enzyme was due to the fact that the purified enzymes were heterodimers formed by the proteolytic processing of the mature enzyme. The derived amino acid sequence of the enzyme showed 30-78% sequence identity with that of other aspartic proteinases.
Park
H
H
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
Yamanaka
N
N
Mikkonen
A
A
Kusakabe
I
I
Kobayashi
H
H
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D006368:Helianthus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D010436:Pepstatins / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D011487:Protein Conformation; D012639:Seeds / Q000201:enzymology* / Q000254:growth & development; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D010436:Pepstatins; D011480:Protease Inhibitors; C014195:Streptomyces pepsin inhibitor; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin