Proteolytic enzymes have many physiological functions, ranging from generalized protein digestion to more specific regulated processes such as the activation of zymogens, blood coagulation and the lysis of fibrin clots, the release of hormones and pharmacologically active peptides from precursor proteins, and the transport of secretory proteins across membranes. They are present in all forms of living organisms. Comparisons of amino acid sequences, three-dimensional structures, and enzymatic reaction mechanisms of proteases indicate that there are distinct families of these proteins. Changes in molecular structure and function have accompanied the evolution of proteolytic enzymes and their inhibitors, each having relatively simple roles in primitive organisms and more diverse and more complex functions in higher organisms.

Many enzymes, hormones, and other physiologically active proteins are synthesized as inactive precursors (zymogens) that are subsequently converted to the active form by the selective enzymatic cleavage (limited proteolysis) of peptide bonds. The ultimate agency of activating enzymatic function is limited proteolysis, either in a single activation step or in a consecutive series (cascade). The specificity of each activation reaction is determined by the complementarity of the zymogen substrate and the active site of the attacking protease. The sequence of consecutive activation reactions is regulated by the specificity of each enzyme, whereas the degree of amplification of the initial stimulus is determined by the efficiency of each activating step.Zymogen activation produces a prompt and irreversible response to a physiological stimulus, and is capable of initiating new physiological functions. Typical examples are the processes of blood coagulation, fibrinolysis, complement activation, hormone production, metamorphosis, rtilization, supramolecular assembly, and digestion. The zymogens of the pancreatic serine proteases, in particular, have served as models for detailed studies of the nature of the molecular changes that are involved in the dramatic increase in enzymatic activity that ensues upon limited proteolysis of the zymogen. In recent years, it has become evident that many proteins are synthesized as inactive precursors or zymogens and that these are subsequently converted to physiologically active forms by the selective enzymatic cleavage of peptide bonds. This process is known as zymogen activation, a term which initially was applied to the activation of precursors of proteolytic enzymes such as trypsinogen, chymotrypsinogen, or procarboxypeptidase (1). It is now apparent that the same type of reaction is involved in a great variety of biological processes, such as blood coagulation, fibrinolysis, complement reaction, hormone production, development, differentiation, and supramolecular assembly, all of which involve zymogen activation in one or more steps (2-9). In the present article, we shall attempt to show that activation by limited proteolysis is indeed an important control element which can initiate new physiological functions or regulate preexistent ones.Virtually all zymogen activation reactions require the en- The position of zymogen activation in the overall scheme of physiological control processes is diagrammatically shown in Fig.

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...

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