Porcine purple acid phosphatase: heterologous expression, characterization, and proteolytic analysis.
Uteroferrin is an iron-binding glycoprotein, which is abundantly synthesized in porcine uterine glandular endometrium and believed to be involved in maternal/fetal iron transport. In the present study, uteroferrin has been cloned and functionally expressed using baculovirus-infected insect host cells Spodoptera frugiperda. The work also addresses the possible role of proteolytic cleavage to facilitate the release of uteroferrin-bound iron. The enzyme secreted in culture medium exhibits a molecular mass and catalytic properties similar to native porcine uteroferrin. The specific activity was estimated at 233 U/mg using p-nitrophenyl phosphate as substrate. Partial cleavage of the enzyme with trypsin resulted in a 1.7-fold enhancement in specific activity and a two-subunit polypeptide as observed in preparations of most mammalian purple acid phosphatases. Digestion with the aspartic protease pepsin resulted in a 2.5-fold enzyme inactivation correlated with the appearance of low molecular weight polypeptide fragments and the release of enzyme-bound iron.
Naseri
Joseph Itor
JI
Institut für Biochemie, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Strasse 2, 48149 Münster, Germany.
Truong
Ngoc Thanh
NT
Hörentrup
Jens
J
Kuballa
Petric
P
Vogel
Andreas
A
Rompel
Annette
A
Spener
Friedrich
F
Krebs
Bernt
B
D000135:Acid Phosphatase / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006023:Glycoproteins / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007313:Insecta; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007527:Isoenzymes; D008667:Metalloproteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D012313:RNA / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013053:Spectrophotometry; D018411:Spodoptera; D013552:Swine; D000071681:Tartrate-Resistant Acid Phosphatase; D013997:Time Factors; D014162:Transfection; D014466:Ultraviolet Rays
D018076:DNA, Complementary; D004279:DNA, Viral; D006023:Glycoproteins; D007527:Isoenzymes; D008667:Metalloproteins; D010455:Peptides; D011994:Recombinant Proteins; D012313:RNA; D007501:Iron; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D006026:Glycoside Hydrolases