Sequence of Tryptic Cleavages in Porcine Pancreatic Secretorv Inhibitor

Abstract The appearance of sequential forms of inhibitor in a mixture of inhibitor and less than equimolar quantities of porcine trypsin at pH 2.75 has been monitored by polyacrylamide gel electrophoresis. Each of the detected forms has been isolated and identified by its Rf in gel electrophoresis, its composition, and its NH2-terminal and COOH-terminal amino acids. The sequence of the bond cleavages by trypsin during temporary inhibition is Lys 14-Ile 15, followed by Arg 40-Gln 41, Lys 48-Ser 49, and Lys 38-Lys 39. The modified inhibitor (with one cleavage at the reactive site) was active while all other forms were inactive. The second cleavage, Arg 40-Gln 41, is the temporary (inactivating) cleavage. The modifying cleavage of the Lys 14-Ile 15 bond was found to be an obligatory step for the further digestion of the inhibitor. The reversible nature of the reactive site bond cleavage was demonstrated in gel electrophoresis by the examination of separate incubations of virgin and modified inhibitors with trypsin. Starting with either form, a mixture of virgin and modified inhibitor was obtained. The modified inhibitor was susceptible to digestion by α-chymotrypsin.

its composition, and its NH9-terminal and COOH-terminal amino acids.
The sequence of the bond cleavages by trypsin during temporary inhibition is Lys 14-Ile 15, followed by Arg 40-Gln 41, Lys 48-Ser 49, and Lys 38-Lys 39. The modified inhibitor (with one cleavage at the reactive site) was active while all other forms were inactive.
The second cleavage, Arg 40-Gln 41, is the temporary (inactivating) cleavage. The modifying cleavage of the Lys 14-Ile 1.5 bond was found to be an obligatory step for the further digestion of the inhibitor.
The reversible nature of the reactive site bond cleavage was demonstrated in gel electrophoresis by the examination of separate incubations of virgin and modified inhibitors with trypsin. Starting with either form, a mixture of virgin and modified inhibitor was obtained.
The modified inhibitor was susceptible to digestion by oc-chymotrypsin.
Methods of prcparatioll of bovinr mid porcine inhibitors of Kazal's type havr, hw improved (S-i). 'I'hc primary structure of bovine sctrrrtory inhibitor has been elucidated by Grec>nc and co-workers (8-10). I'or(*ine sc>rretory inhibitor has bee11 isolated in two forms, at~tl the strurt,ure of both forms has been cxstnblished (I l-15 A preliminary report of this work has appeared (1).
Preliminary results with :I naturally occurring l)orciric~ dc('r(xtory inhibitor (Form I) shon-cd that the motlifying cleavage tlicl not inactivate (I). I'rcseiit s;tutlies were niltlcrtakcn to test the reversibility of this strp and to decide whether the loss of illhibitory activity was causrtl l)y one cleavage or in atc,ps of grutlrinll~decreasing activity.
We chose direct isolation of intermediate forms :I:: a nwtlrotl of study, and selected Form I I of porcine serrctory inhibitor as a starting material bccausc of its simplicity. n-c shall dcscribt: csperiments which show that the modifyin, 1' rlra~~gc iii 3 tiaturally occurring trypsiii inhibitor does not inactivate, that this cleavage is an obligatory stcxp in trmpornry inhibition, anti that a single (in this cast the SNYM~) clrnl-agc~ illactivates. Ttic protein is then digested further.
l'hc resultitig forms have, I)c,rli isolated and identified. It is also obvious that once thr digestion of inhibitor has started it is much faster at pH 8.0 than at acid pH values.
th& point :kii cspcrimrnt was pcrformcd (Fig. 3) in which complex was I'orrnc~l at pH 3.4 and was allowrd to remail1 at that p11 ulitil about 20% of tryljtic activity had reappeared.
At that tinic oiic-lialf of the saml)lc \vas withdra\vn and readjusted to pII 8.0. .\s svw from Fig. 3 the rate of return of tryptic activity is 111url 1 faster at pl I 8 than at pH 3.4. A practical coriclusion from tlicse czl)erimetits (Figs. 2 mid 3) is that the best chance of preserving tlic iritermecliatc~ forms is at acid pH values (e.g. pII 2.7<5).
AII attempt was then matlc to correlate the observed reappcarancc of tryl)t,ic activit,v with the appearance of intermediate forms of the partially tligestetl iiihibitor.
To this end the comples was formed at pH 2.75 and :~llo~ed to stand at room temprraturc.
-it illtrrvals aliquots were ~vithtlrawn, trypsin was removed by prccil)itwtiorr lvith trichloroacetic acid, and the solution of inhibitor was adjusted to $1 9.5 and subjected to gel electrophorrsis (Fig. 4). Th , ; 1: e oe 5 11 erc stained and scanned in a Gilfortl sI)c(~trol)hotorneter, 1notle1 2400. Unfortunately, uniform staining could not be ascertained ant1 therefore the areas uncter the peaks are oiily roughly proportional to the quantity of material and arc not, comparable from one aliquot to another. At that time about 15$;, of tryptic activity is observed (Fig. 2). The result is suggestivc that the modified form is fully active, the next form inactivc. These implications n-err confirmed by direct isolation of iilteriliediatc forms. L\ large scale experiment n-as performed starting with 122 mg of the virgin inhibitor, Form II. The inhibitor was incubated n-ith less than an equivalent amount of trypsin (388 mg) at pH 2.75 for 75 min. 7'1 ie pII was then adjusted to 1.8 to dissociate the comples and the mixture was passed through a Sephadex G-50 column (Fig. 5). The active peak was collected and the protein chrorr~atograI)iletl OII CXSephades as described in the legend to Fig. 6. The peaks marked I and II contained activity. Encti was collected.
Peak I was identified as virgin inhibitor by disc rlrctrol)horesis and by det'ermination of termini. Peak II was further purified as described in the legend to Fig. 7. The first peak of Fig. 7 contained only traces of activity and represented fragments of the trypsin molecule.
The second peak contained inhibitory activity. Specific activity was essentially rotrstmt across the peak, suggesting a high degree of homogcnrity.
The peak material was collected and lyophilized. Pro. 4. Spectrophotometric tracings at 550 nm of stained polyacrylamide gels containing aliqliots (1OOJ) from the incubation of PST1 (2.5 mg) porcine TPCK-trypsin (4 mg) in 0.2 M sodium citrate buffer at pH 2.75. At the times indicated aliquots were pipett,ed into 10 ~1 of 6 N HCl and 10 ~1 of 25y0 tZrichloroacetic acid were added. The samples were frozen and stored prior to elect,rophoresis.
The aliquots were thawed and the precipitated trypsin removed by centrifugation.
The supernatants were mixed wit,h an equal volume of 4061c srrcrose-0.2 Y Tris, pH 10, and were applied to gel columns which were lo';/, in acrylamide and 0.2cs in M,Ar-methylenebisacrylamide at a pH of 9.5. The anionic buffer used as described in the text provided a spacer gel at pH 7.8 and electrode buffer at pH 8.8. The gels were stained in 1% anline black in 7.5% acetic acid and were destained by leeching in 7.5y0 acetic acid. It n-as incubatctl with 1~s than cquimolar amount trypsin at plI 2.75 for IO hours.
The incubation misture was chromatographed on Scphatlcz CL50 (Fig. 9). The first large pc:lk contained act,ivc, trypsill. 'I'hc material marked Peak I was desalted, lyophilized, and c~llrornatographed on SP-Sephades (Fig.  10). The fraction that appc:lrcd after the rluting buffer w-as changed to 0.1 RI acetate, pII 5.4, n-as itlentifietl as two-hit (C) by the amino acid composition :ulcl by -?jHP and COOII termilli ( Table   III Interestingly, the same is true with respect to chymotryptic digestion. The first step of temporary inhibition conforms to the general scheme established by Laskowski and co-workers (see the re- Fig. 14 shows that the modified inhibitor is rapidly digested view (4)). This step is reversible. We have shown reversibility with cr-chymotrypsin, while the virgin inhibitor is not. by establishing the same pseudoequilibrium regardless of whether Electrophoresis and staining were performed as described in Fig. 4. In the Gel A a small amount of virgin inhibitor was added as a rcfcrence to the modified inhibitor, tube B contains only the modified inhibitor (Fig. 7). Form C is a pool from Fig. 10; Forms D and E are pools from Fig. 11. FIG. 13 (right). Disc electrophoresis of separate reaction mixtures of virgin PST1 (2 mg per ml) and modified PST1 (2 mg per ml) and 1 mole y0 TPCK-trypsin at pH 2.75. Incubation conditions, sampling, and electrophoresis were carried out as described for Fig. 4. Tubes 1 to 4 are reaction mixtures containing virgin PST1 after 1 hour, 24 hours, 52 hours, and 73 hours of incubation. Tubes 1' to 4' are analogous except that they contain the modified inhibitor.
The apparent identity of 4 and 4' indicates that a pseudoequilibrium mixture of virgin and modified forms has been established. formittg a cotnplcs in which the reactive site of the inhibitor is brought into close proximity with the actire site of trypsitt. Ko other approach by the inhibitor molecule is possible because it is still resistant to tryptic dig&ion.
Xith tlte puicwatic secretory inhibitor this is not the cast. The tnodifictl irtliibitor may approach the active site of trypsiti either by it-s rwctive site (1+-s 14-11~ 15) or by t,he tcmltorary site (At-g 40.Gltt 31). The first leads to rcformatioti of the stable ~omplcs, tltc: secottd to digestion of the itrltibitor and formation of a sul)strat,c. l'he new form two-hit (C) is totally inactive.