860
NATURALLY OCCURRING PROTEASE INHIBITORS
[75]
[76] P r o t e i n a s e ( E l a s t a s e ) I n h i b i t o r s f r o m Dog Submandibular Glands
By HANS FRITZ and KARL HOCHSTRASSER The highest concentration of a proteinase inhibitor found so far in animal tissues occurs in the submandibular glands of the dog. 1 Depending on the secretory state of the gland, 1 g of fresh tissue contains up to 5 mg of inhibitor. The inhibitor is excreted into the saliva. 2 Outstanding properties of this inhibitory protein are its double-headed nature3--the two reactive sites are independent from each other--the strong inhibition of pancreatic elastase and subtilisin, besides other trypsinand chymotrypsin-like enzymes, 3 and its structural homology to the pancreatic secretory trypsin inhibitors (cf. below).4
Assay Methods
The following enzymes were employed: bovine trypsin and a-chymotrypsin; porcine plasmin (2.68 Novo units/mg) and subtilisin (crystalline bacterial proteinase, 22.0 Anson trypsin units/g) from Novo Industri A/S; Pronase E (70,000 P U K / g from Streptomyces griseus) and porcine pancreatic elastase (cryst., suspension, 15 E/mg) from E. Merck, Darmstadt; alkaline Aspergillus oryzae protease [3500 PU (pH)/mg protein] from RShm & Haas GmbH, Darmstadt. Known methods were used to estimate the inhibition of trypsin (substrafe: N"-benzoyl-Db-arginine p-nitroanilide, BAPA),5,6 chymotrypsin (substrate: N"-succinyl-L-phenylalanine p-nitroanilide, SUPHEPA),~,7 and plasmin (substrate: BAPA).~,5 1E. Werle, I. Trautschold, H. Haendle, and H. Fritz, Ann. N. Y. Acad. Sci. 146, 464 (1968). 2H. Haendle, M.D. Thesis (Dissertation), Medical Faculty of the University of Munich, Munich, 1969. 3H. Fritz, E. Jaumann, R. Meister, P. Pasqay, K. Hochstrasser, and E. Fink, Proteinase Inhibitors, Proc. Int. Res. Conf., 1st, Munich, 1970, p. 257. 4 K. Hochstrasser and H. Fritz. Hoppe-Seyler's Z. Physiol. Chem., 356, 1659 and 1859 (1975). K. Hochstrasser, G. Bretzel, E. Wachter, and S. Heindl, HoppeSeyler's Z. Physiol. Chem. 356, 1865 (1975). H. Fritz, I. Trautschold, and E. Werle, in "Methoden der Enzymatischen Analyse" (I-I. U. Bergmeyer, ed.), 3rd ed., Vol. I, p. 1105. Verlag Chemie, Weinheim, 1974. e B. Kassell, tl~is series Vol. 19, p. 845. 7H. Schiessler, E. Fink, and H. Fritz, this volume [75].
[76]
INHIBITORS
FROM
DOG SUBMANDIBULAR
GLANDS
861
Inhibition of Subtilisin, Aspergillus oryzae Protease, and Pronase. Proteinase activity and enzyme inhibition are measured with azocasein (Pentex-PP from Fluka AG) as substrate. Constant amounts of the enzymes (25/~g of subtilisin, 25 ~g of A. oryzae protease, 50 ~g of pronase) are incubated with increasing amounts of inhibitor (0-15 ~g) in 1.0 ml of 0.1 M sodium potassium phosphate, pH 7.6, for 5 min at 30 °. Subsequently, 2.0 ml of azocasein solution (2%, w/v) in the same buffer are added and the mixture is incubated for 10 rain at 30 °. The enzymic reaction is stopped by the addition of 3.0 ml of aqueous trichloroacetic acid (5%, w/v). After 30 min at room temperature, the extinction at 366 nm of the supernatant is read against a blank sample. The assay method has been described in detail, s Inhibition o] Elastase. The activity of elastase is measured according to Sachar et al. s with elastin-orcein (E. Merck Darmstadt) as substrafe. Elastase inhibition is determined in the following manner: A mixture of 0.15 ml of the elastase suspension (containing about 0.75 mg of elastase in 0.2 M Tris-HC1, pH 8.8) and the inhibitor (0-15 ~g) solution is filled up to 1.50 ml with 0.2 M Tris-HC1, pH 8.8. This incubation mixture is briefly (5 min) shaken and mixed with 20 mg of elastin-orcein. The test sample is vigorously shaken for 30 min at room temperature. The enzymic reaction is stopped by the addition of 2.0 ml of 0.5 M phosphate buffer, pH 6.0. After centrifugation, the extinction at 578 nm of the supernatant is read against a blank sample. One inhibitor unit corresponds to the reduction of the enzymecatalyzed hydrolysis of the substrate by 1 ~mole/min. The inhibitor units given throughout refer to the inhibition of bovine trypsin, applying BAPA as substrate2 The titration curves with the other enzymes are given by Fritz et al) Purification Procedure Dog submandibular glands are extracted either with water or with diluted perchloric acid to minimize proteolytic degradation. Working temperature during the extractions (step la and lb) is 00-4 ° throughout. Step la. Extraction with Water. Dog submandibular glands (Pel Freeze Biologicals, USA) containing 10-14 IU per gram of tissue, are thawed and homogenized in deionized water, 2 liters per 100 g of tissue. After centrifugation the supernatant is adjusted to pH 6.0-6.5 and mixed with 100 g of CM-cellulose (H + form), the suspension being stirred for 2 hr. The CM-cellulose adsorbent is washed three times, each with 500 L. A. Saehar, K. K. Winter, M. Sieher, and S. Frankel, Proc. Soc. Exp. Biol. Med. 90, 323 (1955).
862
NATURALLY OCCURRING PROTEASE INHIBITORS
[7•]
ml of 0.01 M sodium acetate, pH 5.0. Subsequently, the inhibitors are eluted by suspending the adsorbent in 0.01 M triethanolamine (TRA)HC1, 5% (w/v) NaC1, pH 8.0, for 10 rain, several times. Of the inhibitory activity found in the homogenate, 90-95% is recovered in the combined eluates. The eluent is desaltcd by dialysis against deionized water, 4 hr. Concentration either by ultrafiltration (Amicon UM-2 menibrane) or evaporation in vacuo is followed by gel filtration on Sephadex G-50 columns equilibrated and developed with 5% (v/v) acetic acid. Lyophilization of the salt-free inhibitor fractions yields a white powder with a specific activity of 1.4-1.8 IU/mg. About 18% of the inhibitory activity is lost during these procedures. Step lb. Extraction with Perchloric or Formic Acid. The glands are either homogenized directly in 3% (w/v) perchloric acid or the aqueous extract (cf. step la) is diluted with the same volume of 6% pcrchloric acid. The precipitate is removed by centrifugation and extracted three times with 3% perchloric acid. The combined acidic extracts are neutralized by the addition of crystalline K,CO3 (or 5 M K~CO3 solution). Precipitated KCIO~ is removed by filtration. The inhibitor solution thus obtained is diluted with five times its volume of water before CMcellulose is added. Continue as described in step la. If less than 80% of the inhibitory activity is adsorbed to CM-cellulose, the salt concentration of the inhibitor solution has to be reduced either by dialysis or ultrafiltration. Adsorption of the inhibitors to precipitated proteins may be minimized if the glands are homogenized in 3% (v/v) formic acid. After centrifugation, the supernatant is concentrated by evaporation in vacuo or ultrafiltration and fractionated on Sephadex G-75 in 3% (v/v) formic acid. The inhibitor-containing fractions of the eluate are lyophilized. The material is directly applied to step 2a "equilibrium run." Step 2a. Chromatography on CM-Cellulose; Inhibitors ]rom Aqueous Extracts. EQUILIBRIUMRUN. A 2 X 30 cm CM-cellulose column is equilibrated and developed with 0.01 M TRA-HC1, 0.05 M NaC1, pH 8.5, at a rate of 12 ml/hr, 3.5 ml/tube. The solution of 100-150 mg inhibitor from step la in 5 ml of the elution buffer is applied to the column. The inhibitor activity appears in 3 fractions in the eluate (cf. Fig. 1) in the following distribution: 14 ~ 50 = 64% in fractions A1 -~- A_~,7% in fraction B and 28% in fraction C ; 99% in total. GRADIENT RUN. If the inhibitors from step la are separated by gradient elution chromatography as described in step 2b, but with a modified gradient (0.05 M NaC1 in the starting buffer and 0.5 M NaC1 in the second buffer), the inhibitor activity is eluted in two major fractions, about
[76]
INHIBITORS
FROM
DOG S U B M A N D I B U L A R GLANDS
863
50% in fraction I and 30% in fraction II (cf. Fig. 2), the residual activity in intermediate fractions; total, 97%. The inhibitor fractions are desalted by repeated ultrafiltration (Amicon UM-2 membrane) or gel filtration on Bio-Gel P-2 in 0.01 M acetic acid followed by lyophilization. Step 3a. Rechromatography. INHIBITORS A~ AND I FROM STEP 2a. For complete separation of impurities from inhibitor A2 (cf. Fig. 1), fraction A~ from the equilibrium run or fraction I from the gradient run are rechromatographed on a 2 ,~ 65 cm CM-cellulose column equilibrated and developed with 0.01 M TRA-HC1, 0.05 M Nat1, pH 8.5 at a rate of 6 ml/hr, a 2 ml/tube. About 93% of the inhibitory activity applied (230 IU) is thus eluted in a single fraction A~ (symmetrical absorption and activity peak) shortly after the contamination which contains the residual activity (cf. fraction A1 in Fig. 1). Part of the inhibitor activity present in fraction A1 from step 2a can be separated also from the contamination under the same conditions. Inhibitor fraction A._, is desalted by repeated ultrafiltration (Amicon UM-2 membrane), preferably by gel filtration on Sephadex G-50 equilibrated and developed with 5% (v/v) acetic acid followed by lyophilization. A specific activity of 2.3 I U / m g is estimated for inhibitor As. INHIBITOR II FROM STEP 2a. Inhibitor II (gradient run) from step 2a is rechromatographed under the conditions of the equilibrium run of step 2a. From 255 IU applied, 8% of the inhibitory activity eluted in frac-
4-
~3"~,
r
u
r
c
I.
T253
I-
1 _ 1
A
.......
40
I
80
120
Tube no
FIG. 1. Equilibrium chromatography of the dog submandibular inhibitors on CMcellulose (step 2a, equilibrium run). Tubes were combined to yield the fractions as indicated.--, relative transmission at 253 nm (rel. %53).
864
[76]
NATURALLY OCCURRING PROTEASE INHIBITORS
tion A2; in fraction B, 27%; and in fraction C, 54%; in total, 95%. The inhibitor fractions are desalted as described above and lyophilized. Specific activities of 2.2 I U / m g are estimated for these inhibitors, too.
Step 2b. Gradient Elution Chromatography; Inhibitors ]rom Acidic Extracts. A 1.6 )< 30 cm CM-cellulose column is equilibrated and developed with 0.01 M TRA-HC1, pH 8.0, at a rate of 10.5 ml/hr, 5 m l / tube. The solution of 100-140 mg of inhibitor from step lb in 2 ml of the starting buffer is applied to the column. As soon as the protein content and the inhibitor activity in the eluate decrease (cf. Fig. 2), the column is developed with a linear gradient formed from 0.5 liters each of the starting buffer and 0.01 M TRA-HC1, 0.3 M NaC1, pH 8.0. The inhibitor activity is eluted in two major fractions--about 35% in fraction I":, 27% in fraction II":', and the residual activity in intermediate fractions; in total, 98%. Inhibitor fractions I ** and II ** are desalted by repeated ultrafiltration or gel filtration, cf. step 2a.
6 E :K •~
"0
4
. m
re l. T 2 53
lb.
u 0
0
0
c-
•"g2 rl-
i=.
(.9
I
0
0
15
30
Tube no FIG. 2. Gradient chromatography of the dog submanibular inhibitors on CMcellulose (step 2b or 2a, gradient run). Tubes were combined to yield the fractions as indicated. --, relative transmission at 253 nm. (rel. T~8).
[7~]
INHIBITORS FROM DOG SUBMANDIBULAR GLANDS
865
Step 8b. Rechromatography. Inhibitors I *~ and II:: from step 2b are rechromatographed under the same conditions except that the slope of the NaCl-gradient is only two-thirds the one used in step 2b. After desalting and lyophilization, the specific activity of both inhibitors I":" and II': is estimated as 2.3 IU/mg. Comments on the Purification Procedure. Removing acid-labile proreins with perchloric acid simplifies tile isolation and prevents enzymic degradation or limited cleavage of the inhibitor molecules. However, owing to strong adsorption of the inhibitors to the precipitated proteins, the extraction has to be repeated several times. Adsorption is minimized if formic acid is used for deproteinization (extraction). Partial deamidation of the inhibitors in the acidic solutions or cleavage of acid-labile bonds cannot be excluded. Therefore the purification procedure should be selected in view of the desired application of the inhibitors.
Properties Amino Acid Compositions and Molecular Weights. The amino acid compositions of the inhibitors obtained from acidic extracts, Y': and II :'~, and from aqueous extracts, A~ and C, are given in Table I. Although inhibitors I": and A~ have identical compositions, they may differ in the degree of amidation and the nmnber of internal peptide bond cleavages. Compared to inhibitor I '~, a glutamine or glutamic acid residue is exchanged by a lysine residue in inhibitor II% Hence, the assumption of the synthesis of one of these inhibitors by a mutated gene seems logical. Inhibitor C is very probably derived from inhibitor IY'~; glycine, the N-terminal residue, and proline (cf. Fig. 3 below) may be split off by exopeptidases during the first purification steps. Remarkably, the molecular weights calculated for these inhibitors (cf. Table I) are about twice as high as those of the pancreatic secretory trypsin inhibitors (Kazal type)'%1° and that of the trypsin-kallikrein inhibitor from bovine organs. 6 For inhibitor A2 a molecular weight of approximately 12,000 was also estimated by gel filtration, and of about 11,900 from sedimentation studies in the ultracentrifuge. 3 Similar values were reported by other authors21 Inhibition Specificity. The inhibitors from dog submandibular glands (DSI) have an unusually broad inhibition spectrum (Table II). The most striking feature is the strong inhibition of enzymes with chymo9 It. Tschesche, Angew. Chem. 86, 21 (1974); Angew. Chem. Int. Ed. 13, 10 (1974). 10L. J. Greene, D. E. Roark, and D. C. Bartelt, Proteinase Inhibitors, Proc. Int. Res. Con]., 2rid (Bayer Syrup. V), Grosse Ledder, 1973, p. 188. ~1For references see Fritz et al3
866
NATURALLY
OCCURRING
PROTEASE
[76]
INHIBITORS
TABLE I AMINO ACID COMPOSITIONS (MOLES/MoLE) AND MOLECULAR WEIGHTS OF DOG SUBMANDIBULAR INHIBITORS ( D S I ) ~
DSI Residue Asp Thr Ser Glu Pro Gly Ala Cys11~ Val Met lie Leu Tyr Phe Lys His Arg Trp Molecular weight
I* and A2
II*
13 7 8 9 6 9 6 12 4 3 5 6 5 4 10 3 5 0 115 12,750
13 7 8 8 6 9 6 12 4 3 5 6 5 4 11 3 5 0 115 12,750
C 13 7 8 8 5 8 6 12 4 3 5 6 5 4 11 3 5 113 12,595
a Experimental values are given by H. Fritz, E. Jaumann, R. Meister, P. Pasqay, K. Hochstrasser, and E. Fink, Proteinase Inhibitors, Proc. Int. Res. Conf., 1st, Munich, 1970, p. 257. t r y p s i n - l i k e s u b s t r a t e s p e c i f i c i t y : e q u i m o l a r complexes w i t h c h y m o t r y p s i n , p a n c r e a t i c e l a s t a s e , a n d s u b t i l i s i n a r e f o r m e d a l m o s t to t h e e q u i v a l e n c e p o i n t in t h e h i g h l y d i l u t e d solutions of t h e t e s t s y s t e m s 3 ind i c a t i n g K i v a l u e s n e a r or below 1 X l 0 -1° m o l e / l i t e r . S i m i l a r s t r o n g inh i b i t i o n of t h e Aspergillus o r y z a e p r o t e a s e a n d of a c a s e i n - s p l i t t i n g p r o t e a s e p r e s e n t in t h e P r o n a s e e m p l o y e d is o b s e r v e d 2 T h e s e i n h i b i t o r s m a y be used, therefore, to c a l c u l a t e t h e n u m b e r of a c t i v e e n z y m e m o l e cules p r e s e n t in e n z y m e p r e p a r a t i o n s on t h e basis of t h e t i t r a t i o n curves (cf. T a b l e 5 of F r i t z et a l 2 ) . F o r c o m p a r i s o n , t h e i n h i b i t i o n s p e c t r u m of t h e t r y p s i n - k a l l i k r e i n inh i b i t o r from b o v i n e o r g a n s is also shown in T a b l e I I . T h e s p e c i f i c i t y of this i n h i b i t o r is d i r e c t e d m a i n l y a g a i n s t t r y p s i n - l i k e p r o t e i n a s e s . R e a c t i v e Sites. T w o i n d e p e n d e n t r e a c t i v e sites are l o c a t e d on the s u r face of t h e dog s u b m a n d i b u l a r i n h i b i t o r ( D S I ) m o l e c u l e s a t such a d i s t a n c e t h a t t h e f o r m a t i o n of t e r n a r y complexes, e.g., w i t h t r y p s i n and
[76]
867
I N H I B I T O R S FROM DOG SUBMANDIBULAR GLANDS
TABLE II INHIBITION SPECIFICITY OF THE DOG SUBMANDIBULAR ~NHIBITOR (DSI) AND THE TRYPSIN-KALLIKREIN INHIBITOR FROM BOVINE ORGANS (TKI)"
Inhibition by Enzyme
Substrateb
DSF
TKI d
Trypsin, bovine Plasmin, porcine Kallikrein, pancreatic Kallikrein, plasma Chymotrypsin, bovine Elastase, pancreatic Subtilisin, Novo AspergiUus oryzae protease Pronase Collagenase
BAPA BAPA BAEE BAEE SUPHEPA Elastin Azocasein Azocasein Azocasein Synthcticg
+ -~-~--+ + T+ + + + + + +* -
-~--~+ -~+ + + + + -+i -
a Strong, + + ; weaker, + ; --, no inhibition. b BAPA, Na-benzoyl-DL-arginine p-nitroanilide; BAEE, Na-benzoyl-L-arginine ethyl ester; SUPHEPA, N"-succinyl-L-phenylalaninep-nitroanilide. c Cf. H. Fritz, E. Jaumann, R. Meister, P. Pasqay, K. Hochstrasser, and E. Fink, Proteinase Inhibilo~s, Proc. Int. Res. Conf., 1st, Munich, 1970. See B. Kassell, this series Vol. 19, p. 845. Probably a chymotrypsin-like component of Pronase. I A trypsinlike component of Pronase (see B. Kassell, this series Vol. 19, p. 845. p-Phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg-OH [E. Wfinsch and H.-G. Heidrich, Hoppe-Seyler's Z. Physiol. Chem. 333, 149 (1963). c h y m o t r y p s i n or subtilisin, is possible3; the i n h i b i t o r s are " d o u b l e h e a d e d " with " n o t o v e r l a p p i n g " reactive centers2 -° If the a r g i n i n e residue at the a n t i t r y p t i c center is modified, only the t r y p s i n - i n h i b i t i n g p r o p e r t y is lost. E x h a u s t i v e m a l e y l a t i o n does n o t decrease the i n h i b i t o r y effect a g a i n s t t r y p s i n a n d c h y m o t r y p s i n ; this m e a n s t h a t the reactive site peptide bonds are n o t cleaved in the isolated inhibitors. 13 I n h i b i t o r s with reactive centers t h a t do n o t overlap a n d s i m i l a r i n h i b i t i o n specificities are also present in egg white (ovomucoids, o v o i n h i b i tots) ,12,~4 soybeans,15 a n d lima beans. TM 1; R. E. Feeney and R. G. Allison (eds.) "Evolutionary Biochemistry of Proteins," p. 199f. Wiley (Interscicnee), New York, 1969. ~'~D. Kowalski, T. R. Leary, :R. E. McKee, R. W. Sealock, D. Wang, and M. Laskowski, Jr., Proteinase Inhibitors, Proc. Int. Res. ConJ., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 311. 1~R. E. Feeney, Proteinase Inhibitors, Proc. Int. Res. ConJ., 1st, Munich, 1970, p. 189. 1~y. Birk and A. Gertler, Proteinase I~hibitors, Proc. I~t. Res. ConJ., 1st, Munich, 1970, p. 142. 16F. C. Stevens, Proteinase Inhibitors, Proc. Int. Res. Conj., 1st, Munich, 1970, p. 149.
868
[76]
NATURALLY OCCURRING PROTEASE INHIBITORS
AI
I0 T h r S e r P r o Gin A r g _GIu_ Ain T h r CYS T h r S e t Glu Val S e r
-
18 Gly CYS P r o _Lvs_* _I!e_ ~
_Ash
-
P r o v_al
-
"x
BI
R~Asx
-
Ain CYS P r o A r~+._Leu His G_~_
-
Pr__oo fin
-
\
CI
CYS S e r A s x T y r Lys Gly Lys Gly S e r Glu ,~
__
Thr
-
lle
-
Gtu T y r S e r A s p Met CYS T h r Met * A s p Tvr._Asx A r g P r o _Le_u T y r X C y S CYS
CI
Phe CYS
Val
~
CYS G_lu A s n S e r
30 T v r T h r fin
CYS
Met
~
CYS GLx A s h
Ser
T v r Th..~.__~r . L y s His Asp T h r Glv j
Set
~
CYS L v s A s h
Ser
T v r A s n . Lvs Olv A s p S e t Glv /
Glv A s p T h r GIv I"
J
CYS CYS 40 %'Ser GIu A s n Lys Lys Arg Gin T h r P r o Val Leu
A~ Bs
~Ala
Cs
%'Asn A l a Val Lys. S e r Arg Gly T h r Ile Phe R:
Gly P r o P r o P r o Ain lie
Phe T h r Leu Asp _Lvs_,Lys Phe Glx Val Arg
Gly Arg Glx Val
?
50 _Ile Gin I~-.s S e r GI)' P r o
CY_~S
Lys Leu Gin A s p T h r A l a
CYS--C~
_Le_u_A l a Lv_s His Gly Glu
CYS
* Reactive site r e s i d u e
FIG. 3. Comparison of the structures of the inhibitors from porcine pancreas and dog submandibular glands. Details are discussed in the text; text footnote 20. A, porcine pancreatic secretory trypsin inhibitor; B, antitryptic half of the dog submandibular inhibitor; C, antichymotryptic half of the dog submandibular inhibitor.
On the basis of the formation of the appropriate ternary complexes (trypsin and chymotrypsin or subtilisin)3,14 and the specificity requirements of the proteinases, 1ms it can be concluded that the chymotrypsinand the subtilisin-reactive site on the DSI molecule are identical; this same center might be also responsible for the inhibition of elastase and AspergiUus oryzae protease. Therefore, the broad inhibition spectra of this inhibitor (DSI) and of the inhibitors from egg white 14 should be mainly caused by the antichymotryptic centers; the inhibition of chymotrypsin by the trypsin-kallikrein inhibitor from bovine organs (cf. Table II) is a special case. Structural Homology. The pancreatic secretory trypsin inhibitors (Kazal type) belong to a homologous class of proteins which are structurally strongly related to each other. 9,'° The amino acid sequence and the location of the disulfide bridges of the porcine pancreatic trypsin inhibitor 9 are shown in Fig. 3. The structural studies on the dog submandibular inhibitors are still in progress (K. Hochstrasser, E. Wachter, and G. Bretzel) ; however, on the basis of the results thus far available, 2° the peptides could be clearly aligned with the known structure of the pancreatic trypsin inhibitor (cf. Fig. 3). Obviously, the DSI molecules are " D. Shotton, Proteinase Inhibitors, Proc. Int. Res. Con]., 1st, Munich, 1970, p. 47. ~8C. S. Wright, J. Mol. Biol. 67, 151 (1972). 19 L. J. Greene and D. C. Bartelt, in "Protides of the Biological Fluids---23rd Colloquium," (H. Peeters, ed.), Pergamon, Oxford, 1976. 2o The positions of some of the residues have still to be confirmed so that some minor changes are possible later on. A dot between two residues indicates that this sequence has thus far not been confirmed by the analysis of overlapping peptides.
[77]
INI-IIBITORS FROM BRONCHIAL MUCUS
869
composed of two covalently linked halves, the antitryptic part B and the antichymotryptic part C, which are structurally related to each other and to the pancreatic inhibitors. The homology in the amino acid sequences of the peptide chains A2, B2, and C2 and in the location of the disulfide bridges 2 and 3 and the reactive site residues (arginine for trypsin inhibition and perhaps methionine for chymotrypsin and subtilisin inhibition) is especially remarkable. Details of the structural studies and further relations to the structures of other known inhibitors will be discussed elsewhere?
Acknowledgment This work was supported by Sonderforschungsbereich 51, Munich (B-3, B-8),
[77]
Proteinase (Elastase) Inhibitors from the Ciliated Membranes of the Human Respiratory Tract
By
KARL HOCHSTRASSER
From the ciliated membranes of the human respiratory tract, two acid-stable proteinase inhibitors are excreted into the mucus. 1-~ The inhibitors inactivate trypsin, chymotrypsin, and Pronase. The outstanding physiologically interesting properties of the inhibitors are the strong inhibition of the neutral proteinases from leukocytes and its double-headed nature. This means that the reactive sites for trypsin and chymotrypsin are independent from each other, similarly to the dog submandibular inhibitor (see this volume [76]). The inhibitors represent 80% of the total antiproteolytic activity of the secretions (20% is caused by the humoral inhibitor al-antitrypsin). During infections the inhibitors are complexed in a high yield by leukocytic proteinases? ,5 From these findings the physiological function of the inhibitors can be decided. Obviously the inhibitors protect the mucous 1K. Hochstrasser, H. Haendle, R. Reichert, and E. Werle, Hoppe-Seyler's Z. Physiol. Chem 352, 954 (1971). K. Hochstrasser, R. Reichert, S. Schwarz, and E. Werle, Hoppe-Seyler's Physiol. Chem. 353, 221 (1972). '~K. Hochstrasser, R. Reichert, S. Schwarz, and E. Werle, Hoppe-Seyler's Z. Physiol. Chem. 354, 923 (1973). 4R Reichert, K. Hochstrasser, and G. Conradi, Pneumonologie 147, 13 (1972). 5 K. Hochstrasser, K. Schorn, B. Rasche, and C. Raffelt, Pneumonologie 152, 15 (1975).
NATURALLY OCCURRING PROTEASE INHIBITORS
[75]
[76] P r o t e i n a s e ( E l a s t a s e ) I n h i b i t o r s f r o m Dog Submandibular Glands
By HANS FRITZ and KARL HOCHSTRASSER The highest concentration of a proteinase inhibitor found so far in animal tissues occurs in the submandibular glands of the dog. 1 Depending on the secretory state of the gland, 1 g of fresh tissue contains up to 5 mg of inhibitor. The inhibitor is excreted into the saliva. 2 Outstanding properties of this inhibitory protein are its double-headed nature3--the two reactive sites are independent from each other--the strong inhibition of pancreatic elastase and subtilisin, besides other trypsinand chymotrypsin-like enzymes, 3 and its structural homology to the pancreatic secretory trypsin inhibitors (cf. below).4
Assay Methods
The following enzymes were employed: bovine trypsin and a-chymotrypsin; porcine plasmin (2.68 Novo units/mg) and subtilisin (crystalline bacterial proteinase, 22.0 Anson trypsin units/g) from Novo Industri A/S; Pronase E (70,000 P U K / g from Streptomyces griseus) and porcine pancreatic elastase (cryst., suspension, 15 E/mg) from E. Merck, Darmstadt; alkaline Aspergillus oryzae protease [3500 PU (pH)/mg protein] from RShm & Haas GmbH, Darmstadt. Known methods were used to estimate the inhibition of trypsin (substrafe: N"-benzoyl-Db-arginine p-nitroanilide, BAPA),5,6 chymotrypsin (substrate: N"-succinyl-L-phenylalanine p-nitroanilide, SUPHEPA),~,7 and plasmin (substrate: BAPA).~,5 1E. Werle, I. Trautschold, H. Haendle, and H. Fritz, Ann. N. Y. Acad. Sci. 146, 464 (1968). 2H. Haendle, M.D. Thesis (Dissertation), Medical Faculty of the University of Munich, Munich, 1969. 3H. Fritz, E. Jaumann, R. Meister, P. Pasqay, K. Hochstrasser, and E. Fink, Proteinase Inhibitors, Proc. Int. Res. Conf., 1st, Munich, 1970, p. 257. 4 K. Hochstrasser and H. Fritz. Hoppe-Seyler's Z. Physiol. Chem., 356, 1659 and 1859 (1975). K. Hochstrasser, G. Bretzel, E. Wachter, and S. Heindl, HoppeSeyler's Z. Physiol. Chem. 356, 1865 (1975). H. Fritz, I. Trautschold, and E. Werle, in "Methoden der Enzymatischen Analyse" (I-I. U. Bergmeyer, ed.), 3rd ed., Vol. I, p. 1105. Verlag Chemie, Weinheim, 1974. e B. Kassell, tl~is series Vol. 19, p. 845. 7H. Schiessler, E. Fink, and H. Fritz, this volume [75].
[76]
INHIBITORS
FROM
DOG SUBMANDIBULAR
GLANDS
861
Inhibition of Subtilisin, Aspergillus oryzae Protease, and Pronase. Proteinase activity and enzyme inhibition are measured with azocasein (Pentex-PP from Fluka AG) as substrate. Constant amounts of the enzymes (25/~g of subtilisin, 25 ~g of A. oryzae protease, 50 ~g of pronase) are incubated with increasing amounts of inhibitor (0-15 ~g) in 1.0 ml of 0.1 M sodium potassium phosphate, pH 7.6, for 5 min at 30 °. Subsequently, 2.0 ml of azocasein solution (2%, w/v) in the same buffer are added and the mixture is incubated for 10 rain at 30 °. The enzymic reaction is stopped by the addition of 3.0 ml of aqueous trichloroacetic acid (5%, w/v). After 30 min at room temperature, the extinction at 366 nm of the supernatant is read against a blank sample. The assay method has been described in detail, s Inhibition o] Elastase. The activity of elastase is measured according to Sachar et al. s with elastin-orcein (E. Merck Darmstadt) as substrafe. Elastase inhibition is determined in the following manner: A mixture of 0.15 ml of the elastase suspension (containing about 0.75 mg of elastase in 0.2 M Tris-HC1, pH 8.8) and the inhibitor (0-15 ~g) solution is filled up to 1.50 ml with 0.2 M Tris-HC1, pH 8.8. This incubation mixture is briefly (5 min) shaken and mixed with 20 mg of elastin-orcein. The test sample is vigorously shaken for 30 min at room temperature. The enzymic reaction is stopped by the addition of 2.0 ml of 0.5 M phosphate buffer, pH 6.0. After centrifugation, the extinction at 578 nm of the supernatant is read against a blank sample. One inhibitor unit corresponds to the reduction of the enzymecatalyzed hydrolysis of the substrate by 1 ~mole/min. The inhibitor units given throughout refer to the inhibition of bovine trypsin, applying BAPA as substrate2 The titration curves with the other enzymes are given by Fritz et al) Purification Procedure Dog submandibular glands are extracted either with water or with diluted perchloric acid to minimize proteolytic degradation. Working temperature during the extractions (step la and lb) is 00-4 ° throughout. Step la. Extraction with Water. Dog submandibular glands (Pel Freeze Biologicals, USA) containing 10-14 IU per gram of tissue, are thawed and homogenized in deionized water, 2 liters per 100 g of tissue. After centrifugation the supernatant is adjusted to pH 6.0-6.5 and mixed with 100 g of CM-cellulose (H + form), the suspension being stirred for 2 hr. The CM-cellulose adsorbent is washed three times, each with 500 L. A. Saehar, K. K. Winter, M. Sieher, and S. Frankel, Proc. Soc. Exp. Biol. Med. 90, 323 (1955).
862
NATURALLY OCCURRING PROTEASE INHIBITORS
[7•]
ml of 0.01 M sodium acetate, pH 5.0. Subsequently, the inhibitors are eluted by suspending the adsorbent in 0.01 M triethanolamine (TRA)HC1, 5% (w/v) NaC1, pH 8.0, for 10 rain, several times. Of the inhibitory activity found in the homogenate, 90-95% is recovered in the combined eluates. The eluent is desaltcd by dialysis against deionized water, 4 hr. Concentration either by ultrafiltration (Amicon UM-2 menibrane) or evaporation in vacuo is followed by gel filtration on Sephadex G-50 columns equilibrated and developed with 5% (v/v) acetic acid. Lyophilization of the salt-free inhibitor fractions yields a white powder with a specific activity of 1.4-1.8 IU/mg. About 18% of the inhibitory activity is lost during these procedures. Step lb. Extraction with Perchloric or Formic Acid. The glands are either homogenized directly in 3% (w/v) perchloric acid or the aqueous extract (cf. step la) is diluted with the same volume of 6% pcrchloric acid. The precipitate is removed by centrifugation and extracted three times with 3% perchloric acid. The combined acidic extracts are neutralized by the addition of crystalline K,CO3 (or 5 M K~CO3 solution). Precipitated KCIO~ is removed by filtration. The inhibitor solution thus obtained is diluted with five times its volume of water before CMcellulose is added. Continue as described in step la. If less than 80% of the inhibitory activity is adsorbed to CM-cellulose, the salt concentration of the inhibitor solution has to be reduced either by dialysis or ultrafiltration. Adsorption of the inhibitors to precipitated proteins may be minimized if the glands are homogenized in 3% (v/v) formic acid. After centrifugation, the supernatant is concentrated by evaporation in vacuo or ultrafiltration and fractionated on Sephadex G-75 in 3% (v/v) formic acid. The inhibitor-containing fractions of the eluate are lyophilized. The material is directly applied to step 2a "equilibrium run." Step 2a. Chromatography on CM-Cellulose; Inhibitors ]rom Aqueous Extracts. EQUILIBRIUMRUN. A 2 X 30 cm CM-cellulose column is equilibrated and developed with 0.01 M TRA-HC1, 0.05 M NaC1, pH 8.5, at a rate of 12 ml/hr, 3.5 ml/tube. The solution of 100-150 mg inhibitor from step la in 5 ml of the elution buffer is applied to the column. The inhibitor activity appears in 3 fractions in the eluate (cf. Fig. 1) in the following distribution: 14 ~ 50 = 64% in fractions A1 -~- A_~,7% in fraction B and 28% in fraction C ; 99% in total. GRADIENT RUN. If the inhibitors from step la are separated by gradient elution chromatography as described in step 2b, but with a modified gradient (0.05 M NaC1 in the starting buffer and 0.5 M NaC1 in the second buffer), the inhibitor activity is eluted in two major fractions, about
[76]
INHIBITORS
FROM
DOG S U B M A N D I B U L A R GLANDS
863
50% in fraction I and 30% in fraction II (cf. Fig. 2), the residual activity in intermediate fractions; total, 97%. The inhibitor fractions are desalted by repeated ultrafiltration (Amicon UM-2 membrane) or gel filtration on Bio-Gel P-2 in 0.01 M acetic acid followed by lyophilization. Step 3a. Rechromatography. INHIBITORS A~ AND I FROM STEP 2a. For complete separation of impurities from inhibitor A2 (cf. Fig. 1), fraction A~ from the equilibrium run or fraction I from the gradient run are rechromatographed on a 2 ,~ 65 cm CM-cellulose column equilibrated and developed with 0.01 M TRA-HC1, 0.05 M Nat1, pH 8.5 at a rate of 6 ml/hr, a 2 ml/tube. About 93% of the inhibitory activity applied (230 IU) is thus eluted in a single fraction A~ (symmetrical absorption and activity peak) shortly after the contamination which contains the residual activity (cf. fraction A1 in Fig. 1). Part of the inhibitor activity present in fraction A1 from step 2a can be separated also from the contamination under the same conditions. Inhibitor fraction A._, is desalted by repeated ultrafiltration (Amicon UM-2 membrane), preferably by gel filtration on Sephadex G-50 equilibrated and developed with 5% (v/v) acetic acid followed by lyophilization. A specific activity of 2.3 I U / m g is estimated for inhibitor As. INHIBITOR II FROM STEP 2a. Inhibitor II (gradient run) from step 2a is rechromatographed under the conditions of the equilibrium run of step 2a. From 255 IU applied, 8% of the inhibitory activity eluted in frac-
4-
~3"~,
r
u
r
c
I.
T253
I-
1 _ 1
A
.......
40
I
80
120
Tube no
FIG. 1. Equilibrium chromatography of the dog submandibular inhibitors on CMcellulose (step 2a, equilibrium run). Tubes were combined to yield the fractions as indicated.--, relative transmission at 253 nm (rel. %53).
864
[76]
NATURALLY OCCURRING PROTEASE INHIBITORS
tion A2; in fraction B, 27%; and in fraction C, 54%; in total, 95%. The inhibitor fractions are desalted as described above and lyophilized. Specific activities of 2.2 I U / m g are estimated for these inhibitors, too.
Step 2b. Gradient Elution Chromatography; Inhibitors ]rom Acidic Extracts. A 1.6 )< 30 cm CM-cellulose column is equilibrated and developed with 0.01 M TRA-HC1, pH 8.0, at a rate of 10.5 ml/hr, 5 m l / tube. The solution of 100-140 mg of inhibitor from step lb in 2 ml of the starting buffer is applied to the column. As soon as the protein content and the inhibitor activity in the eluate decrease (cf. Fig. 2), the column is developed with a linear gradient formed from 0.5 liters each of the starting buffer and 0.01 M TRA-HC1, 0.3 M NaC1, pH 8.0. The inhibitor activity is eluted in two major fractions--about 35% in fraction I":, 27% in fraction II":', and the residual activity in intermediate fractions; in total, 98%. Inhibitor fractions I ** and II ** are desalted by repeated ultrafiltration or gel filtration, cf. step 2a.
6 E :K •~
"0
4
. m
re l. T 2 53
lb.
u 0
0
0
c-
•"g2 rl-
i=.
(.9
I
0
0
15
30
Tube no FIG. 2. Gradient chromatography of the dog submanibular inhibitors on CMcellulose (step 2b or 2a, gradient run). Tubes were combined to yield the fractions as indicated. --, relative transmission at 253 nm. (rel. T~8).
[7~]
INHIBITORS FROM DOG SUBMANDIBULAR GLANDS
865
Step 8b. Rechromatography. Inhibitors I *~ and II:: from step 2b are rechromatographed under the same conditions except that the slope of the NaCl-gradient is only two-thirds the one used in step 2b. After desalting and lyophilization, the specific activity of both inhibitors I":" and II': is estimated as 2.3 IU/mg. Comments on the Purification Procedure. Removing acid-labile proreins with perchloric acid simplifies tile isolation and prevents enzymic degradation or limited cleavage of the inhibitor molecules. However, owing to strong adsorption of the inhibitors to the precipitated proteins, the extraction has to be repeated several times. Adsorption is minimized if formic acid is used for deproteinization (extraction). Partial deamidation of the inhibitors in the acidic solutions or cleavage of acid-labile bonds cannot be excluded. Therefore the purification procedure should be selected in view of the desired application of the inhibitors.
Properties Amino Acid Compositions and Molecular Weights. The amino acid compositions of the inhibitors obtained from acidic extracts, Y': and II :'~, and from aqueous extracts, A~ and C, are given in Table I. Although inhibitors I": and A~ have identical compositions, they may differ in the degree of amidation and the nmnber of internal peptide bond cleavages. Compared to inhibitor I '~, a glutamine or glutamic acid residue is exchanged by a lysine residue in inhibitor II% Hence, the assumption of the synthesis of one of these inhibitors by a mutated gene seems logical. Inhibitor C is very probably derived from inhibitor IY'~; glycine, the N-terminal residue, and proline (cf. Fig. 3 below) may be split off by exopeptidases during the first purification steps. Remarkably, the molecular weights calculated for these inhibitors (cf. Table I) are about twice as high as those of the pancreatic secretory trypsin inhibitors (Kazal type)'%1° and that of the trypsin-kallikrein inhibitor from bovine organs. 6 For inhibitor A2 a molecular weight of approximately 12,000 was also estimated by gel filtration, and of about 11,900 from sedimentation studies in the ultracentrifuge. 3 Similar values were reported by other authors21 Inhibition Specificity. The inhibitors from dog submandibular glands (DSI) have an unusually broad inhibition spectrum (Table II). The most striking feature is the strong inhibition of enzymes with chymo9 It. Tschesche, Angew. Chem. 86, 21 (1974); Angew. Chem. Int. Ed. 13, 10 (1974). 10L. J. Greene, D. E. Roark, and D. C. Bartelt, Proteinase Inhibitors, Proc. Int. Res. Con]., 2rid (Bayer Syrup. V), Grosse Ledder, 1973, p. 188. ~1For references see Fritz et al3
866
NATURALLY
OCCURRING
PROTEASE
[76]
INHIBITORS
TABLE I AMINO ACID COMPOSITIONS (MOLES/MoLE) AND MOLECULAR WEIGHTS OF DOG SUBMANDIBULAR INHIBITORS ( D S I ) ~
DSI Residue Asp Thr Ser Glu Pro Gly Ala Cys11~ Val Met lie Leu Tyr Phe Lys His Arg Trp Molecular weight
I* and A2
II*
13 7 8 9 6 9 6 12 4 3 5 6 5 4 10 3 5 0 115 12,750
13 7 8 8 6 9 6 12 4 3 5 6 5 4 11 3 5 0 115 12,750
C 13 7 8 8 5 8 6 12 4 3 5 6 5 4 11 3 5 113 12,595
a Experimental values are given by H. Fritz, E. Jaumann, R. Meister, P. Pasqay, K. Hochstrasser, and E. Fink, Proteinase Inhibitors, Proc. Int. Res. Conf., 1st, Munich, 1970, p. 257. t r y p s i n - l i k e s u b s t r a t e s p e c i f i c i t y : e q u i m o l a r complexes w i t h c h y m o t r y p s i n , p a n c r e a t i c e l a s t a s e , a n d s u b t i l i s i n a r e f o r m e d a l m o s t to t h e e q u i v a l e n c e p o i n t in t h e h i g h l y d i l u t e d solutions of t h e t e s t s y s t e m s 3 ind i c a t i n g K i v a l u e s n e a r or below 1 X l 0 -1° m o l e / l i t e r . S i m i l a r s t r o n g inh i b i t i o n of t h e Aspergillus o r y z a e p r o t e a s e a n d of a c a s e i n - s p l i t t i n g p r o t e a s e p r e s e n t in t h e P r o n a s e e m p l o y e d is o b s e r v e d 2 T h e s e i n h i b i t o r s m a y be used, therefore, to c a l c u l a t e t h e n u m b e r of a c t i v e e n z y m e m o l e cules p r e s e n t in e n z y m e p r e p a r a t i o n s on t h e basis of t h e t i t r a t i o n curves (cf. T a b l e 5 of F r i t z et a l 2 ) . F o r c o m p a r i s o n , t h e i n h i b i t i o n s p e c t r u m of t h e t r y p s i n - k a l l i k r e i n inh i b i t o r from b o v i n e o r g a n s is also shown in T a b l e I I . T h e s p e c i f i c i t y of this i n h i b i t o r is d i r e c t e d m a i n l y a g a i n s t t r y p s i n - l i k e p r o t e i n a s e s . R e a c t i v e Sites. T w o i n d e p e n d e n t r e a c t i v e sites are l o c a t e d on the s u r face of t h e dog s u b m a n d i b u l a r i n h i b i t o r ( D S I ) m o l e c u l e s a t such a d i s t a n c e t h a t t h e f o r m a t i o n of t e r n a r y complexes, e.g., w i t h t r y p s i n and
[76]
867
I N H I B I T O R S FROM DOG SUBMANDIBULAR GLANDS
TABLE II INHIBITION SPECIFICITY OF THE DOG SUBMANDIBULAR ~NHIBITOR (DSI) AND THE TRYPSIN-KALLIKREIN INHIBITOR FROM BOVINE ORGANS (TKI)"
Inhibition by Enzyme
Substrateb
DSF
TKI d
Trypsin, bovine Plasmin, porcine Kallikrein, pancreatic Kallikrein, plasma Chymotrypsin, bovine Elastase, pancreatic Subtilisin, Novo AspergiUus oryzae protease Pronase Collagenase
BAPA BAPA BAEE BAEE SUPHEPA Elastin Azocasein Azocasein Azocasein Synthcticg
+ -~-~--+ + T+ + + + + + +* -
-~--~+ -~+ + + + + -+i -
a Strong, + + ; weaker, + ; --, no inhibition. b BAPA, Na-benzoyl-DL-arginine p-nitroanilide; BAEE, Na-benzoyl-L-arginine ethyl ester; SUPHEPA, N"-succinyl-L-phenylalaninep-nitroanilide. c Cf. H. Fritz, E. Jaumann, R. Meister, P. Pasqay, K. Hochstrasser, and E. Fink, Proteinase Inhibilo~s, Proc. Int. Res. Conf., 1st, Munich, 1970. See B. Kassell, this series Vol. 19, p. 845. Probably a chymotrypsin-like component of Pronase. I A trypsinlike component of Pronase (see B. Kassell, this series Vol. 19, p. 845. p-Phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg-OH [E. Wfinsch and H.-G. Heidrich, Hoppe-Seyler's Z. Physiol. Chem. 333, 149 (1963). c h y m o t r y p s i n or subtilisin, is possible3; the i n h i b i t o r s are " d o u b l e h e a d e d " with " n o t o v e r l a p p i n g " reactive centers2 -° If the a r g i n i n e residue at the a n t i t r y p t i c center is modified, only the t r y p s i n - i n h i b i t i n g p r o p e r t y is lost. E x h a u s t i v e m a l e y l a t i o n does n o t decrease the i n h i b i t o r y effect a g a i n s t t r y p s i n a n d c h y m o t r y p s i n ; this m e a n s t h a t the reactive site peptide bonds are n o t cleaved in the isolated inhibitors. 13 I n h i b i t o r s with reactive centers t h a t do n o t overlap a n d s i m i l a r i n h i b i t i o n specificities are also present in egg white (ovomucoids, o v o i n h i b i tots) ,12,~4 soybeans,15 a n d lima beans. TM 1; R. E. Feeney and R. G. Allison (eds.) "Evolutionary Biochemistry of Proteins," p. 199f. Wiley (Interscicnee), New York, 1969. ~'~D. Kowalski, T. R. Leary, :R. E. McKee, R. W. Sealock, D. Wang, and M. Laskowski, Jr., Proteinase Inhibitors, Proc. Int. Res. ConJ., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 311. 1~R. E. Feeney, Proteinase Inhibitors, Proc. Int. Res. ConJ., 1st, Munich, 1970, p. 189. 1~y. Birk and A. Gertler, Proteinase I~hibitors, Proc. I~t. Res. ConJ., 1st, Munich, 1970, p. 142. 16F. C. Stevens, Proteinase Inhibitors, Proc. Int. Res. Conj., 1st, Munich, 1970, p. 149.
868
[76]
NATURALLY OCCURRING PROTEASE INHIBITORS
AI
I0 T h r S e r P r o Gin A r g _GIu_ Ain T h r CYS T h r S e t Glu Val S e r
-
18 Gly CYS P r o _Lvs_* _I!e_ ~
_Ash
-
P r o v_al
-
"x
BI
R~Asx
-
Ain CYS P r o A r~+._Leu His G_~_
-
Pr__oo fin
-
\
CI
CYS S e r A s x T y r Lys Gly Lys Gly S e r Glu ,~
__
Thr
-
lle
-
Gtu T y r S e r A s p Met CYS T h r Met * A s p Tvr._Asx A r g P r o _Le_u T y r X C y S CYS
CI
Phe CYS
Val
~
CYS G_lu A s n S e r
30 T v r T h r fin
CYS
Met
~
CYS GLx A s h
Ser
T v r Th..~.__~r . L y s His Asp T h r Glv j
Set
~
CYS L v s A s h
Ser
T v r A s n . Lvs Olv A s p S e t Glv /
Glv A s p T h r GIv I"
J
CYS CYS 40 %'Ser GIu A s n Lys Lys Arg Gin T h r P r o Val Leu
A~ Bs
~Ala
Cs
%'Asn A l a Val Lys. S e r Arg Gly T h r Ile Phe R:
Gly P r o P r o P r o Ain lie
Phe T h r Leu Asp _Lvs_,Lys Phe Glx Val Arg
Gly Arg Glx Val
?
50 _Ile Gin I~-.s S e r GI)' P r o
CY_~S
Lys Leu Gin A s p T h r A l a
CYS--C~
_Le_u_A l a Lv_s His Gly Glu
CYS
* Reactive site r e s i d u e
FIG. 3. Comparison of the structures of the inhibitors from porcine pancreas and dog submandibular glands. Details are discussed in the text; text footnote 20. A, porcine pancreatic secretory trypsin inhibitor; B, antitryptic half of the dog submandibular inhibitor; C, antichymotryptic half of the dog submandibular inhibitor.
On the basis of the formation of the appropriate ternary complexes (trypsin and chymotrypsin or subtilisin)3,14 and the specificity requirements of the proteinases, 1ms it can be concluded that the chymotrypsinand the subtilisin-reactive site on the DSI molecule are identical; this same center might be also responsible for the inhibition of elastase and AspergiUus oryzae protease. Therefore, the broad inhibition spectra of this inhibitor (DSI) and of the inhibitors from egg white 14 should be mainly caused by the antichymotryptic centers; the inhibition of chymotrypsin by the trypsin-kallikrein inhibitor from bovine organs (cf. Table II) is a special case. Structural Homology. The pancreatic secretory trypsin inhibitors (Kazal type) belong to a homologous class of proteins which are structurally strongly related to each other. 9,'° The amino acid sequence and the location of the disulfide bridges of the porcine pancreatic trypsin inhibitor 9 are shown in Fig. 3. The structural studies on the dog submandibular inhibitors are still in progress (K. Hochstrasser, E. Wachter, and G. Bretzel) ; however, on the basis of the results thus far available, 2° the peptides could be clearly aligned with the known structure of the pancreatic trypsin inhibitor (cf. Fig. 3). Obviously, the DSI molecules are " D. Shotton, Proteinase Inhibitors, Proc. Int. Res. Con]., 1st, Munich, 1970, p. 47. ~8C. S. Wright, J. Mol. Biol. 67, 151 (1972). 19 L. J. Greene and D. C. Bartelt, in "Protides of the Biological Fluids---23rd Colloquium," (H. Peeters, ed.), Pergamon, Oxford, 1976. 2o The positions of some of the residues have still to be confirmed so that some minor changes are possible later on. A dot between two residues indicates that this sequence has thus far not been confirmed by the analysis of overlapping peptides.
[77]
INI-IIBITORS FROM BRONCHIAL MUCUS
869
composed of two covalently linked halves, the antitryptic part B and the antichymotryptic part C, which are structurally related to each other and to the pancreatic inhibitors. The homology in the amino acid sequences of the peptide chains A2, B2, and C2 and in the location of the disulfide bridges 2 and 3 and the reactive site residues (arginine for trypsin inhibition and perhaps methionine for chymotrypsin and subtilisin inhibition) is especially remarkable. Details of the structural studies and further relations to the structures of other known inhibitors will be discussed elsewhere?
Acknowledgment This work was supported by Sonderforschungsbereich 51, Munich (B-3, B-8),
[77]
Proteinase (Elastase) Inhibitors from the Ciliated Membranes of the Human Respiratory Tract
By
KARL HOCHSTRASSER
From the ciliated membranes of the human respiratory tract, two acid-stable proteinase inhibitors are excreted into the mucus. 1-~ The inhibitors inactivate trypsin, chymotrypsin, and Pronase. The outstanding physiologically interesting properties of the inhibitors are the strong inhibition of the neutral proteinases from leukocytes and its double-headed nature. This means that the reactive sites for trypsin and chymotrypsin are independent from each other, similarly to the dog submandibular inhibitor (see this volume [76]). The inhibitors represent 80% of the total antiproteolytic activity of the secretions (20% is caused by the humoral inhibitor al-antitrypsin). During infections the inhibitors are complexed in a high yield by leukocytic proteinases? ,5 From these findings the physiological function of the inhibitors can be decided. Obviously the inhibitors protect the mucous 1K. Hochstrasser, H. Haendle, R. Reichert, and E. Werle, Hoppe-Seyler's Z. Physiol. Chem 352, 954 (1971). K. Hochstrasser, R. Reichert, S. Schwarz, and E. Werle, Hoppe-Seyler's Physiol. Chem. 353, 221 (1972). '~K. Hochstrasser, R. Reichert, S. Schwarz, and E. Werle, Hoppe-Seyler's Z. Physiol. Chem. 354, 923 (1973). 4R Reichert, K. Hochstrasser, and G. Conradi, Pneumonologie 147, 13 (1972). 5 K. Hochstrasser, K. Schorn, B. Rasche, and C. Raffelt, Pneumonologie 152, 15 (1975).
