A peptidase in human platelets that deamidates tachykinins. Probable identity with the lysosomal "protective protein".
We discovered an enzyme in human platelets that deamidates substance P and other tachykinins. Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase. The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94. The purified enzyme exhibits esterase, peptidase, and deamidase activities. The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0. With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5. Enkephalins, released upon deamidation of enkephalinamides, were not cleaved. Gly9-NH2 of oxytocin was released without deamidation. Peptides with a penultimate Arg residue were not hydrolyzed. Some properties of the deamidase are similar to those reported for cathepsin A. The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective. In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer. After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain. The first 25 amino acids of both chains were sequenced. They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast. This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability. A defect in this protein is the cause of galactosialidosis, a severe genetic disorder. The ability of physiological stimuli (e.g. thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.
Laboratory of Peptide Research, University of Illinois College of Medicine, Chicago 60612.
H L
HL
Jackman
F L
FL
Tan
H
H
Tamei
C
C
Beurling-Harbury
X Y
XY
Li
R A
RA
Skidgel
E G
EG
Erdös
D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000378:metabolism; D001792:Blood Platelets / Q000201:enzymology*; D002845:Chromatography; D004600:Eledoisin / Q000378:metabolism; D004950:Esterases / Q000037:antagonists & inhibitors / Q000097:blood; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D008247:Lysosomes / Q000032:analysis* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015288:Neurokinin A / Q000378:metabolism; D010447:Peptide Hydrolases / Q000097:blood*; D011480:Protease Inhibitors / Q000494:pharmacology; D013373:Substance P / Q000378:metabolism; D013379:Substrate Specificity; D015320:Tachykinins / Q000378:metabolism*
D011480:Protease Inhibitors; D015320:Tachykinins; D007531:Isoflurophate; D013373:Substance P; D004600:Eledoisin; D015288:Neurokinin A; D000803:Angiotensin I; D004950:Esterases; D010447:Peptide Hydrolases