Human prothrombin, factor IX, and factor X have been idolated in high yield and characterized as the their amino-terminal sequence, molecular weight, amino acid composition, and migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional human plasma protein, called protein S, has also been purified and its properties have been compared with those of prothrombin, factor IX, and factor X. Prothrombin (mol wt 72 000), factor IX (mol wt 57 000), and protein S (mol wt 69 000) are single-chain glycoproteins, while factor X (mol wt 59 000) is a glycoprotein composed of two polypeptide chains held together by a disulfide bond(s). The amino-terminal sequence of the light chain of human factor X is homologous with prothrombin, factor IX, and protein S. The heavy chain of human factor X is slightly larger than the heavy chain of bovine factor X and differs from bovine factor X in its amino-terminal sequence.

The method of sequenator analysis described by Edman and Begg (Edman, P., and Begg, G. (1967), Eur. J . Biochem. 1, 80) has been modified and applied to proteins and protein fragments. Significant modifications include the replacement of Quadrol by a volatile buffer (dimethylbenzylamine), the introduction of thiols to stabilize the reaction products, and the identification of the reaction products as silylated phenylthiohydantoins by automated gas-liquid chromatography. With these and other modifications, 30-50 T he sequential degradation of peptides by the method of Edman (1956) is an important procedure for the determination of amino acid sequences of proteins. The method combines the specificity of end-group analysis with the advantages of a cyclic stepwise process and normally yields 7-15 unambiguous degradations. In 1967, Edman and Begg automated the process by designing an instrument called the "sequenator" and demonstrated its successful application to the identification of 60 amino-terminal residues of apomyoglobin. Since then, other sequenators have been constructed, built on the principles of Edman and Begg. According to published accounts, these instruments are capable of Smithies et a/., Titani et al., 1972a).The capability of the sequenator to determine long amino acid sequences has altered the general strategy of sequence analysis. Instead of fragmenting the protein into a large number of short peptides whose sequences can be determined by manual Edman degradations and by digestion with carboxypeptidases, the protein is cleaved into a small number of large fragments, usually by chemical procedures (e.g., cyanogen bromide, hydroxylamine), and the separated fragments are directly subjected to automated sequence analysis. Only those segments which cannot be reached by the sequenator are subsequently analyzed by the classical procedures.Sequenator analysis has also been effective for screening proteins for homology, simply by applying the sequential analysis to the amino-terminal region of the protein or to other regions adjacent to existing or newly created a-amino groups. Such initiation points for consecutive degradations can be established by chemical reactions or by limited enzymatic proteolysis.Because of its sensitivity and the small amount of protein required for but a few turns, sequenator analysis is a rapid and amino acid residues can be identified and recovered with a repetitive yield of approximately 96 %. This modified method has been tested on thermolysin and its cyanogen bromide fragments and found to be reliable in determining amino acid sequences. It has also been applied to porcine trypsin and found to be of use in determining purity, allotypic variants, and internal peptide-bond cleavage. In addition, the chemical nature of protein subunits can be identified by this method.accurate test for protein purity and, inter alia, for determining the number of polypeptide chains in a pure oligomeric protein. The method also has proved useful in following the changes in covalent structure att...

ABSTRACEOsmotin is a cationic protein with a pl2 value >8.2 and accounts for 10 to 12% of total cellular protein (as determined by spectrophotometric measurement of Coomassie stained gels) in NaCl and polyethylene glycol adapted tobacco cells (23). In unstressed unadapted cells, an immunologically related 26 kD protein with a lower pl is also synthesized but is not accumulated under normal growth conditions (23). Osmotin synthesis is regulated by abscisic acid, but its accumulation depends on adjustment of the cells to NaCl or to water stress (24). Others using cells obtained from our laboratory, have confirmed our observation of the accumulation of this protein in salt adapted cells (10, 16). We have isolated osmotin from NaCl adapted tobacco cells in sufficient quantities for further characterization. In this report we describe a simple and rapid procedure for purification oftwo forms ofosmotin, some oftheir properties and localization within the cell, and the occurrence of significant amino acid sequence homology between osmotin, the sweet protein, thaumatin, and two other stress-related proteins.MATERIALS AND METHODS Cell Culture. Cell suspension cultures of tobacco (Nicotiana tabacum L. var Wisconsin-38) adapted to 25 g/L NaCl (S-25 cells) were maintained in our laboratory as described (12). These cells were harvested and stored as dry powders after washing in cold acetone (23

No abstract

No abstract