Identification and characterization of proteins synthesized de novo and secreted by the reproductive tract of the American alligator, Alligator mississippiensis.

The objectives of this study were to identify, characterize and examine differences in proteins synthesized de novo and secreted by different regions of the reproductive tract of the American alligator, Alligator mississippiensis, during three reproductive (vitellogenic, gravid, post-clutch) and one non-reproductive state. After capture, alligators from lakes in north central Florida were anaesthetized, the reproductive tract excised aseptically, the size of any follicle determined, and different functional regions of the tract dissected out and partitioned for explant culture. Analysis of the biosynthetic activity indicated regional variations within the tract, differences among reproductive groups and region by status interactions. When oviductal regions were considered regardless of reproductive status, the greatest incorporation of [3H]Leu into secreted nondialysable macromolecules was by the anterior and posterior infundibulum and oviductal tube compared with the transition zone and the uterus. When status was included, the biosynthetic activity of the anterior and posterior portion of the tract in non-reproductive alligators was not different, whereas that of the posterior region of the reproductive group (vitellogenic, gravid, post-clutch) was significantly lower than that of the anterior region. This finding indicates that regulation of protein synthesis and secretion by the non-reproductive alligator tract is different from that in the tract of the reproductive group. Explant-conditioned media were analysed by one-dimensional and two-dimensional SDS-PAGE and fluorography. Sixteen major proteins in culture media were identified as de novo synthesized, by relative molecular weight, by isoelectric point and by differences in distribution determined for reproductive status and oviductal region. Six proteins were examined by N-terminal amino acid microsequence analysis. On the basis of a 29 amino acid sequence, the major oviductal protein, alligator protein 1 (aP1: M(r) 55,000, basic), found in the infundibulum and tube of vitellogenic alligators, was identical to the major protein isolated from alligator egg albumen. Four proteins (aP4-aP7) were sequenced and shown to be significantly related to immunoglobulin heavy chains from several species. This study demonstrated that a large number of proteins are synthesized de novo and released by the female alligator reproductive tract and that there are biosynthetic activity differences by reproductive status and region. Six proteins have been identified, several of which may be incorporated into alligator egg albumen and some of which appear to be different from proteins found in the egg albumens of other species.

Kinetic mechanism of Escherichia coli asparagine synthetase B.

Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartate, glutamine, and ATP. A combination of kinetic, isotopic-labeling, and stoichiometry studies have been performed to define the nature of nitrogen transfer mediated by AS-B. The results of initial rate studies were consistent with initial binding and hydrolysis of glutamine to glutamate plus enzyme-bound ammonia. The initial velocity results were equally consistent with initial binding of ATP and aspartate prior to glutamine binding. However, product inhibition studies were only consistent with the latter pathway. Moreover, isotope-trapping studies confirmed that the enzyme-ATP-aspartate complex was kinetically competent. Studies using 18O-labeled aspartate were consistent with formation of a beta-aspartyl-AMP intermediate, and stoichiometry studies revealed that 1 equiv of this intermediate formed on the enzyme in the absence of a nitrogen source. Taken together, our results are most consistent with initial formation of beta -aspartyl-AMP intermediate prior to glutamine binding. This sequence leaves open many possibilities for the chemical mechanism of nitrogen transfer.

Identification of cysteine-523 in the aspartate binding site of Escherichia coli asparagine synthetase B.

The site-directed chemical modifier [p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA) inactivates Escherichia coli asparagine synthetase B activity following pseudo-first-order kinetics, with ATP providing specific protection, with a Kd of 12 microM. The 5'-FSBA modification appears to be covalent, even though a nonstoichiometric amount (less than 10%) of radiolabeled 5'-FSBA was associated with a totally inactivated enzyme. However, the inactivation by 5'-FSBA could be reversed upon the addition of dithiothreitol. These results are indicative of 5'-FSBA-induced disulfide bond formation, which requires the presence of at least two cysteine residues in the proximity of the ATP binding site. Identification of the critical cysteine residue was accomplished by sequential replacement of each cysteine in the protein by site-directed mutagenesis. Cys 523 was identified as the key residue involved in the formation of the 5'-FSBA-induced disulfide bond. Detailed kinetic analyses and comparison with similar enzymes, suggest that this cysteine residue, while in close proximity to the ATP binding site, is actually involved in aspartate binding in asparagine synthetase B.

Mutagenesis and chemical rescue indicate residues involved in beta-aspartyl-AMP formation by Escherichia coli asparagine synthetase B.

Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of beta-aspartyl-AMP in the reaction mechanism of Escherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317-330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation beta-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to beta-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis.

Arginine 30 and asparagine 74 have functional roles in the glutamine dependent activities of Escherichia coli asparagine synthetase B.

Although Arg-30, Asn-74, and Asn-79 appear totally conserved throughout the purF glutamine-dependent amidotransferases, their potential roles in catalysis and binding remain unexplored for any member of the enzyme family. Here we report the overexpression, purification, and kinetic characterization of a series of AS-B mutants which allow an examination of the functional roles of these 3 residues in glutamine-dependent nitrogen transfer. While Asn-79 appears to possess no catalytic function in AS-B, site-directed mutagenesis of Asn-74 has implicated this residue as playing a role in catalysis of nitrogen transfer from glutamine. The kinetic properties of the Asn-74 AS-B mutant enzymes appear consistent with both ammonia-mediated nitrogen transfer and two apparently novel mechanistic suggestions for this reaction involving either an oxyanion or imide intermediate (Richards, N. G. J., and Schuster, S. M. (1992) FEBS Lett. 313, 98-102). We also demonstrate that replacement of Arg-30 by an alanine residue yields an AS-B mutant for which the apparent Km for glutamine is increased in the glutamine-dependent synthesis of asparagine. In addition, ATP-dependent stimulation of the glutaminase activity of AS-B is modified or completely eliminated when Arg-30 is replaced by other amino acids. The latter observation may indicate the existence of a molecular switch involving Arg-30 which coordinates the two half-reactions catalyzed by the glutamine-dependent amidotransferases and synthetase domains of cellular AS-B.