The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and flconglycinin degradation products was detected by sodium dodecyl sulfatepolyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and B-conglycinin specific antibodies. The three subunits of fl-conglycinin were preferentially metabolized. Of the three subunits of j-conglycinin, the larger a and a' subunits are rapidly degraded, generating new #-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the a-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of f-conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate-polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino-termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth.During the development ofthe dicot seed on the mother plant, storage molecules, particularly starches, proteins, and triglycerides, are laid down in the seed. These reserves are mobilized during germination and seedling growth to supply the energy and metabolic intermediates needed by the seedling prior to the establishment of photosynthetic autotrophism. In the legume seed a relatively large fraction of these reserves, on a weight basis, is composed of storage proteins.The major legume storage proteins are the globulins legumin and vicilin. In the soybean (Glycine max [L.] Merrill) these proteins are called glycinin and fl-conglycinin, respectively.Much work has been done on the structure, biosynthesis, and genetics of these soybean proteins (15,18,20), as well as on their degradation by mammalian trypsin (10-12). However, essen-'Supported by National Science Foundation grant PCM 8301202. A preliminary account of this investigation has been published as Ref. 29. 71 tially no work has been done on their degradation in vivo during germination and seedling growth. We have previously demonstrated that the Bowman-Birk and Kunitz trypsin inhibitor...

During germination, the content of the major Bowman-Birk proteinase inhibitor (BB-E) in the cotyledons of soybean ( Glycine max IL.I Merrill cv. Fiskeby V) seeds decreases, becoming a minor form by the sixth day of germination. One of the three other minor species (BB-D) of this inhibitor in the dry seed increases to become the major form in six-day cotyledons. The other two minor species (BB-C and BB-F) also appear to decrease during germination, but at a slower rate compared to the original major inhibitor form BB-E. By 13 days of germination, no distinct Bowman-Birk inhibitor species can be discerned in the cotyledons. The content of the major Kunitz proteinase inhibitor (K-B) also decreases during germination, but much more slowly. One new form of the Kunitz inhibitor (K-A) becomes apparent by the sixth day. By the 13th day, the proportion of the main isoinhibitor to the late-appearing form is approximately two to one. This difference in the rates of disappearance and appearance of isoinhibitor species in the Bowman-Birk and Kunitz proteinase inhibitor classes suggests a differential metabolism of these two types of proteins and a possible difference in function in the soybean plant.The seeds of legume species contain proteinase inhibitors active against serine proteinases. A number of possible functions for these inhibitors in plants have been proposed. These include the control of endogenous proteinases, particularly during dormancy of the seed, acting as a defense against the proteolytic enzymes of microbial, insect, avian, or mammalian predators, and serving as a storage depot, particularly of sulfur-containing amino acids (15,16). The in vivo function of these proteinase inhibitors is not yet known. In some instances it seems likely that several of these functions may be served simultaneously. A possible storage function is implied by the observed decline in trypsin inhibitory activity during the germination of Vigna radiata (10), Phaseolus vulgaris (14, 18), and Pisum sativum (6). During the germination of the mung bean, V. radiata, the major proteinase inhibitor species present in the dry seed was found to be converted to three new active isoinhibitors (10). Phaseolus angularis proteinase inhibitors have also been shown to undergo limited proteolysis during the germination of the seed (19). mM phenylmethylsulfonylfluoride and 0.5 mm sodium iodoacetate, using a ratio of 10 ml buffer for every gram of cotyledons. The bries were filtered through cheesecloth, then centrifuged at 6,800g at 4°C for 45 min. The supernatants were recentrifuged under the same conditions. Solid ammonium sulfate was then added at 0°C to reach 85% saturation and the material held overnight at 4°C.The ammonium sulfate precipitates were collected the next day by centrifugation at 6,000g at 4°C for 1 h. The remaining pellets were reextracted with buffer, centrifuged, and added to the first extractions. These supernatants were acidified with HCI to a pH of 4.2, the precipitates removed by centrifugation at 6,800g for 1 h and...

The evolutionary history of isozymes for 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase has been constructed in a phylogenetic cluster of procaryotes (superfamily B) that includes Escherichia coli.Members of superfamily B that have been positioned on a phylogenetic tree by oligonucleotide cataloging possess one or more of four distinct isozynies of DAHP synthase. DAHP synthase-0 is insensitive to feedback inhibition, while DAHP synthase-Tyr, DAHP synthase-Trp, and DAHP synthase-Phe are sensitive to feedback inhibition by L-tyrosine, L-tryptophan, and L-phenylalanine, respectively. The evolutionary history of this isozyme family can be deduced within superfamily B by using a cladistic methodology of maximum parsimony (R. A. Jensen, Mol. Biol. Evol. 2:92-108, 1985). DAHP synthase-0 was found in Aeinetobacter species and in Oceanospirillum minutulum, organisms that also possess DAHP synthase-Tyr. These two isozymes were apparently present in a common ancestor that predated the evolutionary divergence of contemporary superfamily B sublineages. DAHP synthase-0 is postulated to have been the evolutionary forerunner of DAHP synthase-Trp. The newly evolved DAHP synthase-Trp is postulated to have possessed sensitivity to feedback inhibition by chorismate as well as by L-tryptophan, chorismate sensitivity having been retained in rRNA group I pseudomonads (minor sensitivity), group V pseudomonads (very sensitive), and Lysobacter enz,ymogenes (ultrasensitive). Organisms constituting the enteric lineage of the phylogenetic tree (including a cluster of four Oceanospirilum species) have all lost the chorismate sensitivity of DAHP synthase-Trp. The absence of DAHP synthase-Phe in the Oceanospirillum cluster of organisms supports the previous conclusion that DAHP synthase-Phe evolved recently within superfamily B, being present only in Escherichia coli and its close relatives.Ever since advancement of the idea (28) that macromolecules might be reliable documents of evolutionary history, a progression of techniques for analytical comparison of nucleic acids has been under development for the purpose of grouping related bacteria. These have included guanine and cytosine contents of DNA (2), DNA-DNA hybridization (9, 10) and DNA-rRNA hybridization (18). An historical and broadly based perspective on the impact of these techniques upon the classification of pseudomonad bacteria, a large and diverse group of prokaryotes having a classic place within the microbiological literature, can be obtained from references 18 and 24. The advent of oligonucleotide cataloging has led to progress so dramatic that phylogenetic trees, once thought impossible to derive, are being constructed at a rapid pace (11,12,(24)(25)(26)(27).Given the new availability of phylogenetic trees, it is now possible to deduce the evolutionary history of biochemical pathways (16). The biosynthetic pathway for aromatic amino acids is complex and varies widely in nature (5). Variable features include alternative biochemical steps, presence of distinctive iso...

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