MULTIPLE DOMAIN KAZAL INHIBITORS
Abstract
The three domains of chicken ovomucoid have been isolated intact using site-specific limited proteolysis to cleave the connecting peptides between domains. Upon titration to low pH, the native molecule and each domain undergoes a nondenaturation chromophoric blue shift. Non-linear least squares analysis of the U.V. difference spectroscopy data yields a midpoint pH value and Hill coefficient for each transition. Although the midpoint pH values range from 2.64 to 3.01, the Hill coefficients are near unity in all cases. Molar absorbancy values indicate that each domain undergoes the low pH transition to nearly the same extent with an average change of (DELTA)a = -1025, which when summed, account for the total decrease in absorbancy for native chicken ovomucoid. The residues involved in the transition have been tentatively identified (tyrosine and aspartic acid present in homologous positions in each domain). These results indicate that to a first order approximation, there is little domain-domain interaction. It is likely that all Kazal inhibitor domains containing the corresponding Asp and Tyr residues will undergo this transition in a similar manner. The implications of independent domains in multiple domain Kazal inhibitors are discussed. Alligator (Alligator mississippiensis) ovomucoid was precipitated from homogenized eggwhites via the conventional TCA-acetone procedure used for avian ovomucoids. The concentration of 1.6 mg/ml is about six times less than that of ovomucoid in avian eggwhites. Characterization and sequence analysis revealed that this molecule is a glycoprotein ((TURN)30% carbohydrate) containing 242 residues with an apparent molecular weight of 45,000. Alligator ovomucoid is homologous and topologically similar to avian ovomucoids, but consists of four tandem, homologous domains. This is the first example of a Kazal inhibitor containing four domains on a single polypeptide chain. The reactive site sequences of each domain are: Domain I: 22 (DARR) 30 -Phe-Cys-Thr-Lys-Ile-Tyr-Ser-Pro-Val-Cys- Domain II: 84 92 -Ala-Cys-Leu-Glu-Ile-Tyr-Ser-Pro-Val-Cys- Domain III: 145 153 -Ala-Cys-Thr-Pro-Asn-Tyr-Lys-Pro-Val-Cys- Domain IV: 202 210 -Ala-Cys-Thr-Leu-Glu-Tyr-Met-Pro-Ile-Cys- We believe this is the first example of an inhibitor with proline in position P(,1) at the reactive site. Alligator ovomucoid is glycosylated at homologous positions in domains I, II and III. These positions, Asn('34), Asn('96) and Asn('157) have never been observed to be glycosylated in any Kazal inhibitor. A fourth glycan is bound to Asn176 in domain III, a position commonly glycosylated in avian ovomucoids. Domain I presumably is the site for the trypsin inhibitory activity of alligator ovomuoid. Domain IV presumably is the site for the inhibition of chymotryspin, subtilisin and elastase. The inhibitory activity of domains II and III is unknown. Evolutionary analysis of these results indicates that most likely a two domain ancestral inhibitor existed at the time of divergence of aves and crocodilians ((TURN)210 x 10('6) years ago). Thus, the events leading to three, and later four, domains in alligator ovomuocid and to three domains in avian ovomucoids apparently took place independently.
Degree
Ph.D.
Subject Area
Biochemistry
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