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[24] Dogfish pepsinogens
364
THE ACIDIC PROTEASES
[24]
[24] Dogfish Pepsinogens By ESTELLE BAR-ELI and T~RENCE G. MEm~ETT
Four pepsinogens have been separated from the stomach mucosae of the smooth dogfish, Mustelus canis. 1 In order to retain some semblance of order in an already confused terminology, we have continued the nomenclature suggested by Ryle for swine pepsinogens 2'3 using as our standard of comparison the order of elution of the proteins from DEAE-cellulose columns at pH 7.5, i.e., pepsinogens B, D, A, and C, respectively. Using this terminology, we find pepsinogen B has enzymatic activity toward synthetic substrates (e.g., N-carbobenzoxy-L-glutamyl-L-tyrosine) at pH 2 and that the other three, after activation, digest protein substrates. Pepsinogens A and D are similar to each other and differ from pepsinogen C.
Assay Methods The Estimation o/Pepsin Using Hemoglobin as Substrate 4
The pH at which this test is carried out also converts pepsinogen into pepsin; therefore, this assay gives peptic activity of pepsin and potential peptic activity of pepsinogen. Denatured hemoglobin is digested under standard conditions, the undigested hemoglobin is precipitated with trichloroacetic acid, and the amount of acid-soluble digested products is estimated by reading the absorbance at 280 nm. The original Anson 4 procedure has been modified as follows: Reagents
Hemoglobin Substrate Powder (Worthington Biochem. Corp.). A 2% solution in 0.06N HC1 is exhaustively dialyzed against 0.06 N HC1 in order to eliminate material that would contribute to the blank. Trichloroacetic acid, 5% in H20 Procedure. One milliliter of 0.06 N HC1 is added to 0.1 ml of the zymogen or enzyme followed by 5.0 ml of 2% hemoglobin solution and the reaction mixtures allowed to incubate at 37 ° for 5-15 minutes. The conditions 1T. G. Merre~tt, E. Bar-Eli, and H. Van Vunakis, Biochemistry 8, 3696 (1969). 2A. P. Ryle, Biochem. J. 96, 6 (1965). ' A. P. Ryle, this volume [20]. • M, L. Anson, J. Gen. Physiol. 22, 79 (1938).
[24]
DOGFISH PEPSINOGENS
365
convert the zymogens to the active enzymes rapidly and no preincubation in acid is required. The solution is deproteinized by the addition of 5 ml of 5% TCA, incubated a further 10 minutes, filtered, and read at 280 nm. The increase in absorbance at 280 nm of the filtrate was a measure of the enzyme activity, and was referred to a standard curve prepared for purified swine pepsin or pepsinogen. All assays were carried out in duplicate together with blank reactions in which the hemoglobin was added after the addition of TCA.
The Estimation o] Pepsin in the Presence o] Pepsinogen Using the Milk Clot Assay at pH 5.4 This modification of the original Klim substrate 5 was developed in the laboratory of Dr. R. M. Herriott.
Reagents Bordens Evaporated Milk (13-oz can) Acetate buffer, 0.22 M, pH 4.6. 170 ml of the buffer is added to the milk with continuous stirring. The substrate is stable for approximately 1 week if kept refrigerated.
The Estimation o] Peptic Activity with Synthetic Substrates N-Cbz-L-Glu-L-Tyr was used as substrate and the extent of hydrolysis determined by the ninhydrin reaction2 Activity was determined by adding a 0.1 ml sample to a 1.0 ml solution containing 5 micromoles of N-Cbz-L-Glu-L-Tyr in 0.02 M NaC1 at pH 2.0. Incubation was at 37 ° for 15 minutes. All assays were carried out in duplicate, together with duplicate blank reactions in which ninhydrin reagent was added to activated enzyme prior to the addition of substrate. After the color was developed in the usual manner, the mean increment of the optical densities at 570 nm was determined and related to a standard tyrosine calibration curve. One unit of enzyme liberates 1 micromole of tyrosine in 1 minute under standard conditions. Purification Procedures All operations were carried out at 4 ° and at pH 7.5 unless otherwise stated. The protein content of crude mucosal extracts in 0.05M Tris buffer at pH 7.5 varied in different preparations from 4 to 6% of the initial tissue weight. Approximately 4% of the total protein was represented by enzyme(s) that digested the blocked dipeptide, N-Cbz-L-GluR. M. Herriott, J. Gen. Physiol. 21, 501 (1938). 6S. Moore and W. H. Stein, J. Biol. Chem. 211~ 907 (1954).
366
THE ACIDIC
PROTEASES
[24]
T,-Tyr and another 8 ~ by enzymes that readily digested hemoglobin at acid pH. The pepsinogens were extracted from the mucosae by dilute buffer since buffers containing (NH4)~SO, fail to extract all of the pepsinogens. Extraction. Smooth dogfish, Mustelus canis, caught at the Woods Hole Marine Biological Laboratories, were killed by a blow on the head, the stomachs removed, washed, and the mucosae stripped and stored in a deep freeze. In a typical run, the mucosae from 7 dogfish stomachs (210 g, although this weight varied with size of stomach) were partially thawed, and homogenized for 30 seconds with 360 ml of 0.05 M Tris buffer. The homogenate was stirred for 1 hour, passed through gauze, and the filtrate centrifuged at 7500 rpm for 45 minutes. The supernatant was further clarified by ultracentrifugation for 45 minutes at a speed of 35,000 rpm and then dialyzed overnight against 0.03 M Tris. High-speed centrifugation of the extract prior to chromatography was essential in order to maintain satisfactory flow properties with DEAE-cellulose columns. DEAE Chromatography. The pepsinogens were adsorbed batchwise from the dialyzed solution onto 250 ml of DEAE-cellulose type 11, previously equilibrated with buffer. After stirring for 30 minutes, the DEAE-cellulose was collected by centrifugation, washed with 400 ml of buffer, and added to a column of DEAE-cellulose (24 X 5 cm) and elution was started using the same 0.03 M Tris buffer. Five hundred milliliters of buffer were sufficient to elute a very large peak of inactive protein and a gradient of increasing molarity was applied by connecting a cylindrical bottle with 0.5M Tris buffer (2000 ml) with a mixing cylindrical chamber containing the starting buffer (2000 ml). Assays using hemoglobin and N-Cbz-L-GIu-L-Tyr as substrates were performed and the peaks pooled accordingly (Fig. 1). The initial purification by the ion-exchanger eliminated a large peak of inactive protein and yielded three smaller peaks which had no enzymatic activity in the milk clot assay at pH 5.4. At acid pH, however, the protein in the first peak hydrolyzed N-Cbz-L-Glu-L-Tyr while the proteins in the second and third peaks were active against hemoglobin. Pepsinogens D, A, C. After dialysis against 0.03 M Tris, the proenzymes in the second (pepsinogens A and D) and third peaks (pepsinogen C) were purified further by repeating the DEAE-cellulose chromatography twice and it was then that the second peak split into two peaks, both of which were capable of digesting hemoglobin after activation (Figs. 2 and 3). Prior to gel filtration on Sephadex G-100, it was necessary to concentrate the individual pepsinogens contained in a volume of approximately 400 ml by adsorbing them onto a small DEAE-cellulose column
[24]
DOGFISH PEPSINOGENS
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Fraction number
FIO. 1. Chromatography of crude dogfish pepsinogens extracted from 7 stomachs on a DEAE-cellulose column in 0.03 M Tris, pH 7~. Tris gradient from 0.03-05 M applied at tube No. 240. Flow rate was 40 ml/hour and fractions of 14 ml were collected. The bars show the fractions pooled for further purification. (O Q) OD 280 nm; (O O) activity against hemoglobin expressed as increase of OD at 280 nm of digested TCA-soluble material; (X X) ninhydrin assay used to follow digestion of N-Cbz4,-Glu-L-Tyr (OD 570 nm).
(30 X 3 cm) in 0.03M Tris, and eluting with 0.6M Tris buffer. The proenzymes were further concentrated about 10-fold, to a volume of 5 ml by ultrafiltration; each was applied to a Sephadex column by the sucrose density layering method and eluted with 0.1 M Tris buffer. Enzyme assays were performed and the potentially active fractions pooled, concentrated by ultrafiltration, and filtered once again through the Sephadex column. The proenzymes were stored frozen in 0.05 M-0.20 M Tris buffer at pH 7.0-7.5. Compared to purified swine pepsinogen, the potential enzymatic activities for pepsinogen A, D, and C were 3.8, 5.3, and 3.0, respectively, in the hemoglobin assay. In the milk clot assay, the enzymatic activities of pepsinogens A, D, and C after conversion into pepsin were all about 5% that of purified swine pepsin. The pepsins were generated from purified pepsinogens D, A, or C by incubating the precursor at a concentration of approximately 1 mg/ml at 37 °, pH 2.0, for 3 minutes. The temperature was brought down to
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FIo. 2. (A) Rechromatography of pepsinogen A on a DEAE--cellulose column (5 X 36 era) in 0.03M Tris, pH 7,5. The elution was carried out with a linear gradient of 0.03-025M Tris applied at tube No. 1 followed by a gradient of 0.25-0,5 M "Iris applied at tube No. 420. Fraction volume was 10 ml. (B) Rechromatography of the leading edge of pepsinogen A peak from DEAE 2nd (Fig. 2A), on a DEAE-cellulose column (3 X 36 cm) in 0.03 M Tris, pH 7,5. The elution was carried out with a linear gradient of 0.03-025 M Tris followed by a gradient of 0.25-0~5 M Tris. The arrow indicates the application of the second gradient. The first peak represents pepsinogen I), followed by pepsinogen A and pepsinogen C. Fraction volume was 10 ml. ( 0 - - - - 0 ) OD 280 nm; (C) O) activity against hemoglobin.
[24]
DOGFISH
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PEPSINOGENS
369
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l~e. 3(A). Rechromatography of pepsinogen C on a DEAE-cellulose column (3 X 36 cm) in 0~ M Tris, pH 7~5, Tris gradient 0~3-0.6M applied at tube No. 1. (B) Second gel filtration of pepsinogen C on Sephadex G-100 (2 X 90 cm) in 0.1 M Tris, pH 7~5. Fraction volume was 3 ml and flow rate 10 ml/hour. (Q Q) OD 280 nm; (O O) activity against hemoglobin. 0% the pH was adjusted to 4.0, and dialysis was carried out against 0.1 M acetate buffer, pH 4.0, to eliminate small peptides. Passage of the digested mixture through Sephadex G-100 also served to remove small peptides. Under these activating conditions, the pepsinogens were converted completely to pepsin (as estimated by milk clot assay) and loss of the generated pepsins by autocatalytic digestion was not observed. The enzymes were stored in 0.1 M acetate buffer, pH 4.6, in a frozen state. Pepsinogen B. The peak which was active against Cbz-L-Glu-L-Tyr was pooled and purified by a second passage through a DEAE-cellulose type 11 column followed by gel filtration chromatography. Although a single symmetrical peak coincident with activity was obtained, polyacrylamide gel electrophoresis at pH 8.5 and high-speed sedimentation to equilibrium demonstrated the presence of 2 components. Pepsinogen B was applied to a column of DEAE-cellulose type 52 equilibrated in 0.03 M Tris and eluted with a gradient from 0.03-0.25 M Tris. The fractions with potential activity toward the synthetic substrate were pooled and concentrated by ultrafiltration and then applied to a Sephadex G-100 column (Fig. 4). These steps resulted in a 2-fold increase in
370
THe. ACIDIC PROTEASES
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FIG. 4(A). Chromatography of pepsinogen B on a DEAE-52 cellulose column (2 X 25 cm) in 0.03 M Tris, pH 7.5. Tris gradient 0.03 M-O25M, fraction volumes 4.0 ml, and flow rate 16 ml/hour; (B) Gel filtration of fraction numbers 55-95 frcan DEAE-52 on a Sephadex G-100 column (1~5X 100 cm) in 0.1M Tris, pH 7,5. (O------~) OD 280 nm; ( O O ) hemoglobin assay--AOD 280 after 45 minutes incubation; (X X) ninhydrin assay was used to follow digestion of N-Cbz L-Glu L-Tyr (AOD 570).
specific activity. Disc-gel electrophoresis indicated the presence of a single component. The final yield from 10 stomachs was 4 mg of protein. Each milligram of protein contained 17 units of activity toward CbzL-Glu-L-Tyr at pH 2.0 under standard conditions. The enzyme is active with synthetic substrates only at acid pH. The molecular weight and amino acid compositions are similar before and after acidification. It has been difficult, therefore, to obtain conclusive evidence for a precursor to enzyme conversion since properties which could distinguish the two molecules have not yet been found. Two possibilities exist, the conversion may occur rapidly at acid pH and not involve great changes in structure, or what we are calling pepsinogen B is in reality a peptidase active at acid pH. Properties From physical, chemical, and immunochemical data, 1,7 pepsinogen A and D appear to be closely related to each other and different from pepsinogen C. The pepsins generated from the proenzymes also show this similarity, i.e., only pepsin A and D resemble each other. Pepsinogen B appears unrelated to either pepsinogen A and D or pepsinogen C systems. The molecular weights of pepsinogen D, A, and C were found to be approximately 42,000 and their amino acid compositions are shown in T. G. Merrett, L. Levine, and H. Van Vunakis, submitted to Immunochemistry.
[24]
DOGFISH PEPSINOGENS
371
AMINO ACID COMPOSITIONS OF DOGFISH PEPSINOGENS
Dogfish pepsinogens
Lys His Arg Asp Thr Ser Glu Pro Gly Ala Half-Cys Val Met Ile Leu Tyr Phe Trp
A
C
D
14 7 14 44 23 43 39 19 40 18 7 23 7 22 28 19 17 5
5 4 8 44 27 51 41 22 48 18 7 27 6 18 24 20 17 --
11 7 15 45 24 38 38 17 43 16 7 25 7 22 25 21 18 --
the table. The amide phosphate a n d / o r sugar contents have not yet been determined nor has any separate determination been made to estimate relative contents of cysteine/cystine. At p H 8.5 on acrylamide gels, the order of their mobilities toward the anode was C ~ A > D as would be expected from their amino acid compositions and chromatographic properties on D E A E . The pepsins which are generated from the purified precursors have molecular weights of 34,000 to 36,000 by ultracentrifugal analysis, indicating a loss of fragments with molecular weights of approximately 7000 during the conversion process. The molecular weight of pepsinogen B was 96,000 from sedimentation-equilibrium runs and 45,000 from molecular sieve chromatography on a calibrated Sephadex G-100 column. It is more basic than pepsinogens D, A, and C. Stabilities. The dogfish enzymes are more stable than swine pepsin at neutral pH. Lyophilization of both precursors and enzymes should be avoided since it leads to aggregation with loss in potential enzymatic or enzymatic activities. Specificities. Dogfish pepsins A, D, and C are active on protein substrates at acid p H only and show no activity toward the synthetic substrate, N-Cbz-L-Glu-L-Tyr. Pepsin A has a broader pH optimum
372
THE
ACIDIC P R O T E A S E S
[25]
than swine pepsin using hemoglobin as a substrate. Differences in specificities exist since dogfish pepsins A, D, and C digest various protein substrates differently from each other and from swine pepsin. Their specificities on protein substrates of known sequence are unknown. Pepsin B has no activity toward hemoglobin. There is, however, a second pepsinogen B fraction in which the activities toward N-CbzL-Glu-L-Tyr and hemoglobin have proven inseparable by the chromatographic methods used thus far. Pepsin B is active only at acid pH and can hydrolyze the N-Cbz derivatives of L-Tyr-L-Tyr, Gly-L-Tyr, L-Glu-L,Phe, Gly-L-Phe, L-Phe-L-Tyr, L-Tyr-L-Phe, and L-Phe-L-Phe in addition to the test substrate N-Cbz-L-Glu-L-Tyr.
[25] Microbial Acid Proteinases By J. ~ODEK and T. HOFMANS
Introduction The presence of proteolytic enzymes with a p H optimum in the acid p H range (pH I-5) has been reported in a variety of microorganisms where they occur both intracellularly and extracellularly.The enzymes from Eumycota (true fungi) have been studied most extensively and many have been isolated and purified. Acid proteinases are also found in many protozoa, both as intracellularand extracellular enzymes? The intracellular enzyme from Tetrahymena pyri]ormis has been partially purified? On the other hand, there are only few reports of the occurrence of acid proteinases among the bacteria: Several strains of Clostridia (acetobutylicum and butyricum) 3 and lactobacilli ~ have been shown to produce weak proteinase activity with an acid pH optimum. No information appears to be available on acid proteinases in algae. The only enzymes which have been fully purified are of fungal origin and are extracellular. The intracellular acid enzyme (proteinase A) from bakers' yeast was first noted by Dernby 5 and described by Lenney6 and was partially purified by Hata et al/ 1M. Muller, in "Chemical Zoology" (G. W. Kidder, ed.), Vol. I, p. 374. Academic Press, New York, 1967. N. Dickie and I. E. Liener, Biochim. Biophys. Acta 64, 41 (1962). s F. Uchino, K. Miura, and S. Doi, J. Ferment. Technol. 46, 188 (1968). ' V . Bottaszi, Intern. Dairy Congr. Proc. 16th Copenhagen 2, 522 (1962). s K. G. Dernby, Biochem. Z. 81, 107 (1917). 6j. F. Lenney, J. Biol. Chem. 221, 919 (1956).
' T. Hata, R. Hayashi, and E. Doi, Agr. Biol. Chem. 31, 357 (1967).
THE ACIDIC PROTEASES
[24]
[24] Dogfish Pepsinogens By ESTELLE BAR-ELI and T~RENCE G. MEm~ETT
Four pepsinogens have been separated from the stomach mucosae of the smooth dogfish, Mustelus canis. 1 In order to retain some semblance of order in an already confused terminology, we have continued the nomenclature suggested by Ryle for swine pepsinogens 2'3 using as our standard of comparison the order of elution of the proteins from DEAE-cellulose columns at pH 7.5, i.e., pepsinogens B, D, A, and C, respectively. Using this terminology, we find pepsinogen B has enzymatic activity toward synthetic substrates (e.g., N-carbobenzoxy-L-glutamyl-L-tyrosine) at pH 2 and that the other three, after activation, digest protein substrates. Pepsinogens A and D are similar to each other and differ from pepsinogen C.
Assay Methods The Estimation o/Pepsin Using Hemoglobin as Substrate 4
The pH at which this test is carried out also converts pepsinogen into pepsin; therefore, this assay gives peptic activity of pepsin and potential peptic activity of pepsinogen. Denatured hemoglobin is digested under standard conditions, the undigested hemoglobin is precipitated with trichloroacetic acid, and the amount of acid-soluble digested products is estimated by reading the absorbance at 280 nm. The original Anson 4 procedure has been modified as follows: Reagents
Hemoglobin Substrate Powder (Worthington Biochem. Corp.). A 2% solution in 0.06N HC1 is exhaustively dialyzed against 0.06 N HC1 in order to eliminate material that would contribute to the blank. Trichloroacetic acid, 5% in H20 Procedure. One milliliter of 0.06 N HC1 is added to 0.1 ml of the zymogen or enzyme followed by 5.0 ml of 2% hemoglobin solution and the reaction mixtures allowed to incubate at 37 ° for 5-15 minutes. The conditions 1T. G. Merre~tt, E. Bar-Eli, and H. Van Vunakis, Biochemistry 8, 3696 (1969). 2A. P. Ryle, Biochem. J. 96, 6 (1965). ' A. P. Ryle, this volume [20]. • M, L. Anson, J. Gen. Physiol. 22, 79 (1938).
[24]
DOGFISH PEPSINOGENS
365
convert the zymogens to the active enzymes rapidly and no preincubation in acid is required. The solution is deproteinized by the addition of 5 ml of 5% TCA, incubated a further 10 minutes, filtered, and read at 280 nm. The increase in absorbance at 280 nm of the filtrate was a measure of the enzyme activity, and was referred to a standard curve prepared for purified swine pepsin or pepsinogen. All assays were carried out in duplicate together with blank reactions in which the hemoglobin was added after the addition of TCA.
The Estimation o] Pepsin in the Presence o] Pepsinogen Using the Milk Clot Assay at pH 5.4 This modification of the original Klim substrate 5 was developed in the laboratory of Dr. R. M. Herriott.
Reagents Bordens Evaporated Milk (13-oz can) Acetate buffer, 0.22 M, pH 4.6. 170 ml of the buffer is added to the milk with continuous stirring. The substrate is stable for approximately 1 week if kept refrigerated.
The Estimation o] Peptic Activity with Synthetic Substrates N-Cbz-L-Glu-L-Tyr was used as substrate and the extent of hydrolysis determined by the ninhydrin reaction2 Activity was determined by adding a 0.1 ml sample to a 1.0 ml solution containing 5 micromoles of N-Cbz-L-Glu-L-Tyr in 0.02 M NaC1 at pH 2.0. Incubation was at 37 ° for 15 minutes. All assays were carried out in duplicate, together with duplicate blank reactions in which ninhydrin reagent was added to activated enzyme prior to the addition of substrate. After the color was developed in the usual manner, the mean increment of the optical densities at 570 nm was determined and related to a standard tyrosine calibration curve. One unit of enzyme liberates 1 micromole of tyrosine in 1 minute under standard conditions. Purification Procedures All operations were carried out at 4 ° and at pH 7.5 unless otherwise stated. The protein content of crude mucosal extracts in 0.05M Tris buffer at pH 7.5 varied in different preparations from 4 to 6% of the initial tissue weight. Approximately 4% of the total protein was represented by enzyme(s) that digested the blocked dipeptide, N-Cbz-L-GluR. M. Herriott, J. Gen. Physiol. 21, 501 (1938). 6S. Moore and W. H. Stein, J. Biol. Chem. 211~ 907 (1954).
366
THE ACIDIC
PROTEASES
[24]
T,-Tyr and another 8 ~ by enzymes that readily digested hemoglobin at acid pH. The pepsinogens were extracted from the mucosae by dilute buffer since buffers containing (NH4)~SO, fail to extract all of the pepsinogens. Extraction. Smooth dogfish, Mustelus canis, caught at the Woods Hole Marine Biological Laboratories, were killed by a blow on the head, the stomachs removed, washed, and the mucosae stripped and stored in a deep freeze. In a typical run, the mucosae from 7 dogfish stomachs (210 g, although this weight varied with size of stomach) were partially thawed, and homogenized for 30 seconds with 360 ml of 0.05 M Tris buffer. The homogenate was stirred for 1 hour, passed through gauze, and the filtrate centrifuged at 7500 rpm for 45 minutes. The supernatant was further clarified by ultracentrifugation for 45 minutes at a speed of 35,000 rpm and then dialyzed overnight against 0.03 M Tris. High-speed centrifugation of the extract prior to chromatography was essential in order to maintain satisfactory flow properties with DEAE-cellulose columns. DEAE Chromatography. The pepsinogens were adsorbed batchwise from the dialyzed solution onto 250 ml of DEAE-cellulose type 11, previously equilibrated with buffer. After stirring for 30 minutes, the DEAE-cellulose was collected by centrifugation, washed with 400 ml of buffer, and added to a column of DEAE-cellulose (24 X 5 cm) and elution was started using the same 0.03 M Tris buffer. Five hundred milliliters of buffer were sufficient to elute a very large peak of inactive protein and a gradient of increasing molarity was applied by connecting a cylindrical bottle with 0.5M Tris buffer (2000 ml) with a mixing cylindrical chamber containing the starting buffer (2000 ml). Assays using hemoglobin and N-Cbz-L-GIu-L-Tyr as substrates were performed and the peaks pooled accordingly (Fig. 1). The initial purification by the ion-exchanger eliminated a large peak of inactive protein and yielded three smaller peaks which had no enzymatic activity in the milk clot assay at pH 5.4. At acid pH, however, the protein in the first peak hydrolyzed N-Cbz-L-Glu-L-Tyr while the proteins in the second and third peaks were active against hemoglobin. Pepsinogens D, A, C. After dialysis against 0.03 M Tris, the proenzymes in the second (pepsinogens A and D) and third peaks (pepsinogen C) were purified further by repeating the DEAE-cellulose chromatography twice and it was then that the second peak split into two peaks, both of which were capable of digesting hemoglobin after activation (Figs. 2 and 3). Prior to gel filtration on Sephadex G-100, it was necessary to concentrate the individual pepsinogens contained in a volume of approximately 400 ml by adsorbing them onto a small DEAE-cellulose column
[24]
DOGFISH PEPSINOGENS
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367
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560
480
600
Fraction number
FIO. 1. Chromatography of crude dogfish pepsinogens extracted from 7 stomachs on a DEAE-cellulose column in 0.03 M Tris, pH 7~. Tris gradient from 0.03-05 M applied at tube No. 240. Flow rate was 40 ml/hour and fractions of 14 ml were collected. The bars show the fractions pooled for further purification. (O Q) OD 280 nm; (O O) activity against hemoglobin expressed as increase of OD at 280 nm of digested TCA-soluble material; (X X) ninhydrin assay used to follow digestion of N-Cbz4,-Glu-L-Tyr (OD 570 nm).
(30 X 3 cm) in 0.03M Tris, and eluting with 0.6M Tris buffer. The proenzymes were further concentrated about 10-fold, to a volume of 5 ml by ultrafiltration; each was applied to a Sephadex column by the sucrose density layering method and eluted with 0.1 M Tris buffer. Enzyme assays were performed and the potentially active fractions pooled, concentrated by ultrafiltration, and filtered once again through the Sephadex column. The proenzymes were stored frozen in 0.05 M-0.20 M Tris buffer at pH 7.0-7.5. Compared to purified swine pepsinogen, the potential enzymatic activities for pepsinogen A, D, and C were 3.8, 5.3, and 3.0, respectively, in the hemoglobin assay. In the milk clot assay, the enzymatic activities of pepsinogens A, D, and C after conversion into pepsin were all about 5% that of purified swine pepsin. The pepsins were generated from purified pepsinogens D, A, or C by incubating the precursor at a concentration of approximately 1 mg/ml at 37 °, pH 2.0, for 3 minutes. The temperature was brought down to
A
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150
200
250
Fraction number
FIo. 2. (A) Rechromatography of pepsinogen A on a DEAE--cellulose column (5 X 36 era) in 0.03M Tris, pH 7,5. The elution was carried out with a linear gradient of 0.03-025M Tris applied at tube No. 1 followed by a gradient of 0.25-0,5 M "Iris applied at tube No. 420. Fraction volume was 10 ml. (B) Rechromatography of the leading edge of pepsinogen A peak from DEAE 2nd (Fig. 2A), on a DEAE-cellulose column (3 X 36 cm) in 0.03 M Tris, pH 7,5. The elution was carried out with a linear gradient of 0.03-025 M Tris followed by a gradient of 0.25-0~5 M Tris. The arrow indicates the application of the second gradient. The first peak represents pepsinogen I), followed by pepsinogen A and pepsinogen C. Fraction volume was 10 ml. ( 0 - - - - 0 ) OD 280 nm; (C) O) activity against hemoglobin.
[24]
DOGFISH
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PEPSINOGENS
369
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20
z~4o 5o ~o 7o
l~e. 3(A). Rechromatography of pepsinogen C on a DEAE-cellulose column (3 X 36 cm) in 0~ M Tris, pH 7~5, Tris gradient 0~3-0.6M applied at tube No. 1. (B) Second gel filtration of pepsinogen C on Sephadex G-100 (2 X 90 cm) in 0.1 M Tris, pH 7~5. Fraction volume was 3 ml and flow rate 10 ml/hour. (Q Q) OD 280 nm; (O O) activity against hemoglobin. 0% the pH was adjusted to 4.0, and dialysis was carried out against 0.1 M acetate buffer, pH 4.0, to eliminate small peptides. Passage of the digested mixture through Sephadex G-100 also served to remove small peptides. Under these activating conditions, the pepsinogens were converted completely to pepsin (as estimated by milk clot assay) and loss of the generated pepsins by autocatalytic digestion was not observed. The enzymes were stored in 0.1 M acetate buffer, pH 4.6, in a frozen state. Pepsinogen B. The peak which was active against Cbz-L-Glu-L-Tyr was pooled and purified by a second passage through a DEAE-cellulose type 11 column followed by gel filtration chromatography. Although a single symmetrical peak coincident with activity was obtained, polyacrylamide gel electrophoresis at pH 8.5 and high-speed sedimentation to equilibrium demonstrated the presence of 2 components. Pepsinogen B was applied to a column of DEAE-cellulose type 52 equilibrated in 0.03 M Tris and eluted with a gradient from 0.03-0.25 M Tris. The fractions with potential activity toward the synthetic substrate were pooled and concentrated by ultrafiltration and then applied to a Sephadex G-100 column (Fig. 4). These steps resulted in a 2-fold increase in
370
THe. ACIDIC PROTEASES
[241
I B Ii
A 70.8
0.8
2.4
2.0 1.6
1.2 $
0.6 o
0
0.4 0.2
x
/ ~'5
0.4
\.-,_ 50 75 ~00 Fr0cti0n number
to.2 t25
t50
0.2
.4
c~ 0.1
0
20 40 60 80 Fraction number
FIG. 4(A). Chromatography of pepsinogen B on a DEAE-52 cellulose column (2 X 25 cm) in 0.03 M Tris, pH 7.5. Tris gradient 0.03 M-O25M, fraction volumes 4.0 ml, and flow rate 16 ml/hour; (B) Gel filtration of fraction numbers 55-95 frcan DEAE-52 on a Sephadex G-100 column (1~5X 100 cm) in 0.1M Tris, pH 7,5. (O------~) OD 280 nm; ( O O ) hemoglobin assay--AOD 280 after 45 minutes incubation; (X X) ninhydrin assay was used to follow digestion of N-Cbz L-Glu L-Tyr (AOD 570).
specific activity. Disc-gel electrophoresis indicated the presence of a single component. The final yield from 10 stomachs was 4 mg of protein. Each milligram of protein contained 17 units of activity toward CbzL-Glu-L-Tyr at pH 2.0 under standard conditions. The enzyme is active with synthetic substrates only at acid pH. The molecular weight and amino acid compositions are similar before and after acidification. It has been difficult, therefore, to obtain conclusive evidence for a precursor to enzyme conversion since properties which could distinguish the two molecules have not yet been found. Two possibilities exist, the conversion may occur rapidly at acid pH and not involve great changes in structure, or what we are calling pepsinogen B is in reality a peptidase active at acid pH. Properties From physical, chemical, and immunochemical data, 1,7 pepsinogen A and D appear to be closely related to each other and different from pepsinogen C. The pepsins generated from the proenzymes also show this similarity, i.e., only pepsin A and D resemble each other. Pepsinogen B appears unrelated to either pepsinogen A and D or pepsinogen C systems. The molecular weights of pepsinogen D, A, and C were found to be approximately 42,000 and their amino acid compositions are shown in T. G. Merrett, L. Levine, and H. Van Vunakis, submitted to Immunochemistry.
[24]
DOGFISH PEPSINOGENS
371
AMINO ACID COMPOSITIONS OF DOGFISH PEPSINOGENS
Dogfish pepsinogens
Lys His Arg Asp Thr Ser Glu Pro Gly Ala Half-Cys Val Met Ile Leu Tyr Phe Trp
A
C
D
14 7 14 44 23 43 39 19 40 18 7 23 7 22 28 19 17 5
5 4 8 44 27 51 41 22 48 18 7 27 6 18 24 20 17 --
11 7 15 45 24 38 38 17 43 16 7 25 7 22 25 21 18 --
the table. The amide phosphate a n d / o r sugar contents have not yet been determined nor has any separate determination been made to estimate relative contents of cysteine/cystine. At p H 8.5 on acrylamide gels, the order of their mobilities toward the anode was C ~ A > D as would be expected from their amino acid compositions and chromatographic properties on D E A E . The pepsins which are generated from the purified precursors have molecular weights of 34,000 to 36,000 by ultracentrifugal analysis, indicating a loss of fragments with molecular weights of approximately 7000 during the conversion process. The molecular weight of pepsinogen B was 96,000 from sedimentation-equilibrium runs and 45,000 from molecular sieve chromatography on a calibrated Sephadex G-100 column. It is more basic than pepsinogens D, A, and C. Stabilities. The dogfish enzymes are more stable than swine pepsin at neutral pH. Lyophilization of both precursors and enzymes should be avoided since it leads to aggregation with loss in potential enzymatic or enzymatic activities. Specificities. Dogfish pepsins A, D, and C are active on protein substrates at acid p H only and show no activity toward the synthetic substrate, N-Cbz-L-Glu-L-Tyr. Pepsin A has a broader pH optimum
372
THE
ACIDIC P R O T E A S E S
[25]
than swine pepsin using hemoglobin as a substrate. Differences in specificities exist since dogfish pepsins A, D, and C digest various protein substrates differently from each other and from swine pepsin. Their specificities on protein substrates of known sequence are unknown. Pepsin B has no activity toward hemoglobin. There is, however, a second pepsinogen B fraction in which the activities toward N-CbzL-Glu-L-Tyr and hemoglobin have proven inseparable by the chromatographic methods used thus far. Pepsin B is active only at acid pH and can hydrolyze the N-Cbz derivatives of L-Tyr-L-Tyr, Gly-L-Tyr, L-Glu-L,Phe, Gly-L-Phe, L-Phe-L-Tyr, L-Tyr-L-Phe, and L-Phe-L-Phe in addition to the test substrate N-Cbz-L-Glu-L-Tyr.
[25] Microbial Acid Proteinases By J. ~ODEK and T. HOFMANS
Introduction The presence of proteolytic enzymes with a p H optimum in the acid p H range (pH I-5) has been reported in a variety of microorganisms where they occur both intracellularly and extracellularly.The enzymes from Eumycota (true fungi) have been studied most extensively and many have been isolated and purified. Acid proteinases are also found in many protozoa, both as intracellularand extracellular enzymes? The intracellular enzyme from Tetrahymena pyri]ormis has been partially purified? On the other hand, there are only few reports of the occurrence of acid proteinases among the bacteria: Several strains of Clostridia (acetobutylicum and butyricum) 3 and lactobacilli ~ have been shown to produce weak proteinase activity with an acid pH optimum. No information appears to be available on acid proteinases in algae. The only enzymes which have been fully purified are of fungal origin and are extracellular. The intracellular acid enzyme (proteinase A) from bakers' yeast was first noted by Dernby 5 and described by Lenney6 and was partially purified by Hata et al/ 1M. Muller, in "Chemical Zoology" (G. W. Kidder, ed.), Vol. I, p. 374. Academic Press, New York, 1967. N. Dickie and I. E. Liener, Biochim. Biophys. Acta 64, 41 (1962). s F. Uchino, K. Miura, and S. Doi, J. Ferment. Technol. 46, 188 (1968). ' V . Bottaszi, Intern. Dairy Congr. Proc. 16th Copenhagen 2, 522 (1962). s K. G. Dernby, Biochem. Z. 81, 107 (1917). 6j. F. Lenney, J. Biol. Chem. 221, 919 (1956).
' T. Hata, R. Hayashi, and E. Doi, Agr. Biol. Chem. 31, 357 (1967).