We characterized the structure, organization, and expression of genes that encode the soybean glycinins, a family of storage proteins synthesized exclusively in seeds during embryogenesis. Five genes encode the predominant glycinin subunits found in soybeans, and they have each been cloned, sequenced, and compared. The five genes have diverged into two subfamilies that are designated as Group-I and Group-II glycinin genes. Each glycinin gene contains four exons and three introns like genes that encode related proteins in other legumes. Two other genes have been identified and designated as "glycinin-related" because they hybridize weakly with the five glycinin genes. Although not yet characterized, glycinin-related genes could encode other glycinin subunit families whose members accumulate in minor amounts in seeds. The three Group-I glycinin genes are organized into two chromosomal domains, each about 45 kilobase pairs in length. The two domains have a high degree of homology, and contain at least five genes each that are expressed either in embryos or in mature plant leaves. Gel blot studies with embryo mRNA, as well as transcription studies with 32P-RNA synthesized in vitro from purified embryo nuclei, indicate that glycinin and glycinin-related genes become transcriptionally activated in a coordinated fashion early in embryogenesis, and are repressed coordinately late in seed development. In addition to transcriptional control processes, posttranscriptional events also are involved in regulating glycinin and glycinin-related mRNA levels during embryogenesis.

Factor VIIa (VIIa), the serine protease that initiates the coagulation pathways, is catalytically activated upon binding to its cell surface receptor and cofactor tissue factor (TF). This study provides a comprehensive analysis of the functional surface of VIIa by alanine scanning mutagenesis of 112 residues. Residue side chains were defined which contribute to TF binding and factor X hydrolysis. Energetically important binding contacts at the interface with TF were identified in the first epidermal growth factor domain of VIIa (Gln-64, Ile-69, Phe-71, Arg-79) and in the protease domain (Arg-277, Met-306, Asp-309). The observed energetic defects are in good agreement with the corresponding residues in TF, suggesting that the VIIa light chain plays a prominent role in high affinity binding of cofactor. Mutation of protease domain interface residues indicated that TF allosterically inf luences the active site of VIIa. Stabilization of a labile zymogen to enzyme transition could explain the activating effect of TF on VIIa catalytic function. Residues important for factor X hydrolysis were found in three regions of the protease domain: (i) specificity determinants in the catalytic cleft and adjacent loops, (ii) an exosite near the TF binding site, and (iii) a large electronegative exosite which is in a position analogous to the basic exosite I of thrombin. TF regions involved in factor X activation are positioned on the same face of the TF⅐VIIa complex as the two exosites identified on the protease domain surface, providing evidence for an extended interaction of TF⅐VIIa with macromolecular substrate.Coagulation factor VIIa (VIIa) is the initiating protease of the coagulation pathways (1). VIIa binds to its cellular receptor and catalytic cofactor, the transmembrane glycoprotein tissue factor (TF). Interaction of VIIa with TF markedly enhances the catalytic activity of the serine protease which is a poor enzyme when free in solution. However, the mechanism by which the cofactor achieves this activation is unknown. VIIa is a multidomain enzyme characterized by an amino-terminal ␥-carboxyglutamic acid-rich (Gla) domain, two epidermal growth factor (EGF)-like modules and a trypsin-like serine protease domain. Various experimental approaches indicate that the Gla-domain, the first EGF domain (EGF1), and the protease domain provide the most significant contributions to the interaction with TF (reviewed in ref.2). The recently determined three-dimensional structure of the TF⅐VIIa complex revealed a discontinuous interaction involving each domain of VIIa and the two fibronectin type III modules that constitute the cytokine receptor-like extracellular domain of TF (3).While extensive mutagenesis of TF has defined the functional and energetic importance of residues that interface with VIIa, there is little information on the contributions of specific amino acid side chains in VIIa to TF binding or proteolytic function. The following surface-exposed residues in VIIa are known to play a functionally important role: Arg...

No abstract