Determination of disulfide bond pairs and stability in recombinant tick anticoagulant peptide.
Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of blood coagulation factor Xa (Waxman, L., Smith, D.E., Arcuri, K.E., and Vlasuk, G.P. (1990) Science 248, 593-596). The 60-amino acid sequence of TAP shows limited homology to Kunitz-type inhibitors, including cysteines at positions 5, 15, 33, 39, 55, and 59. For detailed biochemical and pharmacological studies, a recombinant version of TAP (rTAP) has been produced in yeast. To determine the arrangement of the disulfide bonds, rTAP was cleaved with trypsin and chymotrypsin and the purified peptides sequenced using a gas-phase sequenator. The positions of the disulfide bonds were assigned by identifying the cycle(s) at which di-phenylthiohydan-toin-cystine was released. The specific disulfide bridges, Cys-5 to Cys-59, Cys-15 to Cys-39, and Cys-33 to Cys-55, are analogous to those in the prototype Kunitz-type inhibitor, bovine pancreatic trypsin inhibitor (BPTI). While treatment of BPTI with dithiothreitol rapidly and specifically reduced one disulfide bond, the reduction of disulfide bonds in rTAP proceeded at a slower rate and appeared to be nonspecific, reaching a maximum of two disulfides reduced. Reduced rTAP derivatized with either iodoacetic acid or iodoacetamide lost 59% of its inhibitory activity. In contrast, BPTI alkylated with iodoacetic acid inhibited trypsin half as well as the iodoacetamide derivative. Although the arrangement of disulfides in the two inhibitors is the same, their susceptibility to reduction is markedly different.
Sardana
M
M
Department of Biological Chemistry, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Sardana
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V
Rodkey
J
J
Wood
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T
Ng
A
A
Vlasuk
G P
GP
Waxman
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L
D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins; D002851:Chromatography, High Pressure Liquid; D003553:Cystine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D065427:Factor Xa Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors / Q000737:chemistry; D013987:Ticks / Q000032:analysis
D060829:Arthropod Proteins; D004220:Disulfides; D065427:Factor Xa Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C063967:tick anticoagulant peptide; D003553:Cystine