PLANT PROTEASES

I. ACTIVATION-INHIBITION REACTIONS

BY DAVID M. GREENBERG AND THEODORE WINNICK

(From the Division of Biochemistry, University of California Medical School, Berkeley)

(Received for publication, May 13, 1940)

The recent work of Balls and Lineweaver (1) with crystalline papain supports the view that one or more sulfhydryl groups are essential to the activity of this enzyme. These investigators have adopted the term papainases to characterize the various plant proteolytic enzymes which resemble papain in their reversible activation and inhibition reactions.

It is commonly stated that the properties of bromelin of pine- apple are the same as those of papain, although no thorough study of the former enzyme has been made. In the present paper, the nature of the active groups of bromelin is investigated with the aid of reagents that have come into recent use for this purpose. In addition, the activation-inhibition reactions of two other pro- teases are described. One of these enzymes is from the horse- nettle, Solanum elaeagnijolium, whose milk-clotting action was first studied by Bodansky (2). The name solanain is suggested for this enzyme.1 The other protease, hitherto unreported, is from the latex of the milkweed, Asclepias mexicana. We have recently reported on the properties of the protease from a different milkweed, Asclepias speciosa (3), and in this paper these enzymes are distinguished by the names asclepain m and asclepain s.

Certain experiments with papain and asclepain s, designed to test the sulfhydryl theory, are also described. It will be shown that bromelin, asclepain m, and asclepain s are papainases, but that solanain gives none of the reactions suggestive of the presence of an active sulfhydryl, and accordingly, the last enzyme is not a papainase.

1 It is suggested by Dr. H. Lineweaver and by us that the ending “ain” be used to form the generic names of new proteases from plant sources.

761

by guest on May 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

762 Plant Proteases. I

Isolation of Proteases

Bromelin--To obtain this enzyme, 3 liters of juice from fresh pineapple fruit2 were filtered with the aid of super-eel, and ad- justed to pH 6 with ammonia. Then solid (NH&S04 was added to the point of saturation. The resulting precipitate of crude enzyme was centrifuged down, collected on a Buchner filter, and washed with 0.6 saturated (NHJ2S04. The precipitate was then redissolved in a liter of 0.02 M NaCN (pH S), and the solution again made 0.6 saturated with (NH4)2S04. The precipitate, col- lected as before, was drained as dry as possible, and redissolved in 600 ml. of 0.02 M NaCN. Then the enzyme was precipitated by the addition of 3 volumes of acetone. It was centrifuged down, collected on a Buchner filter, washed with acetone followed by ether, and finally dried in a vacuum desiccator. The almost colorless, dry product that resulted weighed 5 gm.

Asclepain m-10 ml. of latex from Asclepias mexicana were extracted with 10 ml. of 0.05 M NaCN (pH 7), and the filtrate saturated with solid (NH4)2S04. The resulting precipitate was collected on a filter, washed with 0.7 saturat,ed (NH&S04, drained free of liquid, and then dried in a vacuum desiccator. The light yellow product weighed about 100 mg.

Solanain-This enzyme was prepared from the fruit of Solanum elaeagnijolium.4 Bodansky’s isolation procedure (2) was modified as follows :

The fresh fruit was ground and extracted with dilute phosphat’e buffer (pH 7.5), and the extract centrifuged free of solids. The dark green solution was made up to about 0.7 saturation with solid (NH&S04. The resulting precipitate of crude enzyme was centrifuged down, collected on a Buchner filter, washed with 0.7 saturated (NHJ2S04, and drained dry. The precipitate was

2 Pineapple fruit was kindly sent to us by Dr. J. L. Collins of the Pine- apple Producers Experiment Station of the University of Hawaii.

3 This milkweed, which was found growing wild near Pinole, California, differs greatly from Asclepias speciosa in size an8 structure. It is a small weed, usually growing to less than a foot in height, and it yields but a drop or two of latex per plant.

4 Collections of the fruit were kindly sent to us by Dr. R. Chandler of t,he Botany Department, and Dr. W. H. Brown of the Zoology Department, of the Universit,y of Arizona.

by guest on May 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

D. M. Greenberg and T. Winnick

readily dissolved in water, and the enzyme reprecipitated by adding 4 volumes of acetone. The precipitate was washed with acetone and drained free of liquid. It was again dissolved in water, precipitated by 0.7 saturation with (NH&S04, and col- lected on a filter. Finally, the enzyme preparation was precipi- tated twice more from aqueous solution by 4 volumes of acetone, washed with acetone and ether, and dried in a vacuum desiccator. About 0.8 gm. of white product was the yield per 100 gm. of fresh fruit.

Asclepain s and Papain-These enzymes were prepared as previously described (3).

Methods

The activating or inhibiting effect of different reagents on the proteolytic activity of the enzymes was measured by the same technique used in previous studies with asclepain s. The enzyme solutions were treated with the specified reagents for an hour at room temperature in the cases of asclepain m and bromelin, and for about 12 hours in the experiments with solanain. Then the proteolytic activity of the treated solutions was measured on 2 per cent Van Slyke casein at 40” by the Northrop and Kunitz non-protein nitrogen method (4). In the measurements with asclepain m and bromelin, the casein substrate, buffered at pH 7.5, was digested for 30 minutes. For solanain the substrate was buffered at pH 8.5, and the digestion time was 60 minutes. The original enzyme solutions contained 1.5 mg. of asclepain m, and 3.0 mg. of bromelin or solanain, per ml.

The degree of activation or inhibition is expressed in each case as the ratio of the activity of the treated enzyme to that of the untreated enzyme solution. The activity values, expressed as milliequivalents of non-protein nitrogen produced in 6 ml. of digestion mixture in a definite time, are recorded for the untreated enzymes, but are omitted for all the treated enzyme solutions, and only the ratios are given. Enzyme solutions, when treated with maleic acid, always stood at least 12 hours before further treat- ment.

In certain special experiments, reported in Table III and else- where in the text, proteolytic activity was measured by Anson’s hemoglobin method (5).

by guest on May 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

764 Plant Proteases. I

DISCUSSION

Asclepain m and Bromelin-The results of the activation- inhibition measurements with asclepain m and bromelin are given in Tables I and II. The behavior of these enzymes, in most

TABLE I

Activalion-Znhibition of Asclepain m

The untreated enzyme solution, diluted with 2 volumes of water, pro- duced 0.172 to 0.178 milliequivalent of non-protein nitrogen in 6 ml. of digestion mixture.

Reagent added to enzyme solution

Inhibitor

None “ “

0.005 M Hz02 Same 0.001 M K3Fe(CN)6 Same 0.0005 M I,

Same 0.01 M iodoacetic acid Same 0.03 M maleic acid Same 5 mg. CUZO Same 0.001 N Ag+ or Hg++ 1 X 1lP N Ag+ 3 x 10-6 I( i( 1 x 10-6 (i ci

0.01 N Ag+ Same 0.01 N Hg++ 0.001 “ Ag+ Same

iatio of activity to that of untreated

enzyme Activator - 0.05 M cysteine 0.1 “ NaCN 0.1 ” H,S None 0.05 M cysteine None 0.1 M NaCN None 0.1 M NaCN None 0.05 M cysteine None 0.1 M NaCN None 0.1 M H,S None

I‘ ,‘ ‘I

0.1 M HzS 0.1 “ NaCN 0 1 “ H,S o:l “ I‘

0.1 “ NaCN

2.98 2.78 2.66 0.02 2.70 0.02 2.74 0.06 0.15 0.01 0.00 0.08 2.65 0.04 0.08 0.00-0.02 0.20 0.52 0.82 0.04 1.46 0.48 2.00 2.12

respects, resembles the effects that have been reported for papain and asclepain s.

It has been pointed out by Anson (6) and by Hellerman (7) that sulfhydryl groups with different degrees of reactivity may be present in different native proteins, and sometimes in a single

by guest on May 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

D. M. Greenberg and T. Winnick 765

protein. Balls and Lineweaver (8) likewise conclude that the -SH of native crystalline papain is able to react with certain

TABLE II Activation-Inhibition of Bromelin

The untreated enzyme solution, diluted with 2 volumes of water, pro- duced 0.223 to 0.255 milliequivalent of non-protein nitrogen in 6 ml. of digestion mixture.

Reagent added to eneyme solution II Inhibitor ACtiW&tOr

ietio of activity to that of untreated

CIlZYl3W

None “ ,‘ I‘ ‘C

0.003 M Hz02 Same 0.01 M KaFe(CN)e* Same 0.001 M KMnO* Same 0.001 M 12 Same 0.02 M iodoacetic acid Same 0.03 M maleic acid Same 5 mg. Cu10 Same 0.01 N Ag+ or Hg++ 1 X 1OF N Ag+ 2 x IO-6 (6 I(

0.67 X 10-e N Ag+ 0.01 N Ag+ Same

0.001 N Ag+ or Hg++ 0.001 “ “

0.06 M cysteine 0.03 “ “ 0.1 “ NaCN 0.1 “ HkJ 0.5 ” NaaS None 0.1 M NaCN None 0.1 M NaCN None 0.1 M NaCN None 0.03 M cysteine None 0.1 M NaCN None 0.1 M NaCN None 0.1 M H,S None

I‘ “ ‘I

0.1 M H# 0.5 “ NazS 0.1 “ NaCN 0.1 “ HzS 0.1 “ NaCN

2.28 2.04-2.16 2.15 1.18-1.25 1.10 0.04 0.84 0.60 1.90 0.16 1.18 0.00 0.26 0.01 0.00 1.08 2.18 0.08 0.18 0.00-0.02 0.22 0.62 0.82 0.01-0.04 0.50 1.76-1.90 1.26-1.28 2.12

* This reagent was allowed to act for 12 hours.

reagents but not with others. If it is assumed that reversibly oxidized thiol groups in enzymes can likewise have different re-

by guest on May 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Plant Proteases. I

activities, one has a further basis for interpreting activation- inhibition differences. The responses of the enzymes depend not only upon the oxidation-reduction potentials of the reagents, but upon the particular conditions that determine the reaction rates (9).

Activation by Reducing Agents-In Table I it is seen that ascle- pain m is activated to approximately the same degree by different reducing agents if sufficiently high concentrations are employed. This resembles the finding of Balls and Lineweaver (1) with crystalline papain. It seems likely that cysteine, cyanide, and H$ all cause the same chemical change. To account for this on the basis of the formation of thiol groups in the enzymes, it must be assumed that CN- is oxidized to CNO- and H&S to S.

In the case of bromelin (Table II), cysteine and cyanide activate to about the same degree, while H&S and NazS produce a much lower degree of activation. Rather than postulate a different reaction product in the latter case, as was tentatively done by Hellerman and Perkins (10) for crude papain, it may be simply that the oxidized sulfhydryl groups of bromelin are not readily reducible by sulfides.

Fruton and Bergmann (11) found that papain, activated with cysteine or cyanide, becomes completely inactive to synthetic peptides when precipitated by isopropyl alcohol, and that the enzyme precipitate recovers full activity when again treated with the activators. This led them to favor the older view that these activators form dissociable compounds with papain, and therefore function as coenzymes rather than as reducing agents.

Experiments like those of Fruton and Bergmann were carried out by us on papain and asclepain s. The details are, briefly, as follows :

Preparations of asclepain s and papain were dissolved in 0.05 M neutral cysteine or cyanide solutions. The enzymes were pre- cipitated with 4 volumes of ethyl or isopropyl alcohol, collected on Buchner filters, and washed with absolute alcohol. From the precipitates, solutions were made up that contained equal con- centrations (0.2 mg. per ml.) of each enzyme in both pure water and in 0.05 M HCN (pH 7). The proteolytic activity of these solutions was then measured by Anson’s hemoglobin method, with 10 minute digestions at 30”.

by guest on May 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

D. M. Greenberg and T. Winnick 767

The results in Table III show that asclepain s, activated with cysteine or HCN, loses only 10 per cent of its activity in the above precipitation procedure. Activated papain loses about half its activity when precipitated by ethyl or isopropyl alcohol. When the papain precipitate was strongly aerated, there was a large irreversible loss of activity.

In the opinion of the authors, the reversible losses in activity are probably produced by mild oxidation of the enzymes by oxygen, owing to the presence of catalyzing impurities, and not to dissociation of an enzyme-activator compound. The energetic aeration of the papain precipitate probably oxidized the active

TABLE III

Effect oJ Alcohol Precipitation on Activity of Asclepain s and Papain

i

Enzyme Initial activator

Asclepain s

Papain

Cysteine HCN Cysteine

“ “

Proteolytic activity of redissolved enzyme

(tyrosine in 6 ml. digestion Alcohol used to ppt. mixture)

ensyme NOTI- Enzyme

activated treated with IXGSyIIle HCN

m.eq. x 108 m.eq. x 108

Ethyl 6.7 7.3 ‘L 6.4 7.2 ‘I 3.0 6.6

Isopropyl 3.2 7.1 Ethyl, ppt. aerated 0.45 1.3

30 min. on filter

sulfhydryl groups beyond the reversible stage. It is significant that highly purified papain is stable toward oxygen, and can be salted-out of 70 per cent alcohol by lithium sulfate, and subse- quently recrystallized from water without loss of activity (1). This evidence opposes the view that alcohol dissociates a papain- activator complex.

Reversible Inactivation by Oxidants-When asclepain m is com- pletely inactivated by dilute Hz02 or ferricyanide, 90 to 100 per cent of the full activity can be subsequently restored by the addi- tion of excess cyanide or cysteine. These effects strongly suggest an oxidation-reduction process. An explanation in terms of the dissociable complex theory seems less plausible.

by guest on May 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

768 Plant Proteases. I

When bromelin is inactivated by dilute Hz02 or KMnO+ subse- quent treatment with cyanide does not produce full activation. This suggests that part of the sulfur is oxidized beyond the re- versible stage. The partial inactivation obtained with ferricya- nide (acting for a much longer time) and the subsequent reactivation closely resemble the effects obtained by Hellerman and Perkins with impure papain (10). Apparently, ferricyanide is unable to oxidize all of the sulfhydryl, but, like Hz02 and KMn04, can oxidize part of it past the reversible stage. It may be noted in this connection that the “free” sulfhydryl groups of native egg albumin do not react at all with ferricyanide (6), even at pH 9.6.

Ketene-The irreversible inactivations found with iodine suggest that aromatic groups may be iodinated in these reactions. In connection with this possibility, it was of interest to test the action of ketene on papain.6 This reagent inactivates pepsin presumably by acetylating the phenolic hydroxyl groups in the enzyme (12). The course of the inactivation of papain by ketene was found to resemble that of pepsin. Ketene, acting on a 0.1 per cent solution of papain (activated with HCN) at O”, and buffered at pH 5.5, caused a 20 per cent loss in activity in 5 minutes, and a 70 per cent loss in 1 hour.

Since it seemed possible that ketene might acetylate the -SH groups of papain, this was guarded against in another experiment by mildly oxidizing the enzyme with dilute Hz02 before subjecting it to acetylation. In this experiment the papain lost 55 per cent of its potential activity in 5 minutes, and 95 per cent in 1 hour, upon treatment with ketene.

These results suggest that ketene may react with groups in papain other than sulfhydryl, which are also essential for proteo- lytic activity. The slow rate of the reaction makes it seem unlikely that these are primary amino groups. Balls and Line- weaver (1) likewise believe that the NH2 groups of papain are unrelated to the enzyme activity. Accordingly, in line with the deductions for pepsin, it seems possible that phenolic groups, in addition to -SH, are essential for the proteolytic activity of papain.

Iodoacetic Acid-This reagent, which reacts vigorously with

6 We thank Dr. C. H. Li of the Institute of Experimental Biology of the University of California for the use of his ketene generator.

by guest on May 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

D. M. Greenberg arid T. Winnick 769

-SH compounds, completely inactivates asclepain m and bro- melin. None of the activity is restored by adding an excess of activator. Balls and Lineweaver (8) have shown that the inhibi- tion of papain activity is produced by 1 molecular equivalent of iodoacetate, and have detected hydriodic acid as a product of the reaction, so that the reaction mechanism seems fairly well established.

According to Maschmann (13), the inactivation of papain by iodoacetate is reversed by precipitation of the enzyme with alco- hol. This experiment was repeated by us, using 0.02 M iodo- acetic acid (pH 7) completely to inactivate a 0.3 per cent papain solution. Following alcohol precipitation, we found no recovery in the proteolytic activity of the enzyme.

Cuprous O&e-This substance, which reversibly inactivates papain at pH 5 (lo), inactivates asclepain m and bromelin irre- versibly at pH 7. The nature of the reaction here is not known.

Maleic Acid-Morgan and Friedmann (14) found that the incubation of papain with maleic acid at 37” and at pH 4.7 pro- duced 70 per cent inhibition of the enzyme after 16 hours. By analogy with the reactions of thiol compounds, these workers conclude that maleic acid probably forms an addition compound with reduced papain. No attempt was made to reverse the inactivation.

We have obtained 90 per cent inhibition of papain with maleic acid at pH 7.0 by incubation for 5 hours at 37”. This inactivation was about 70 per cent reversed by the subsequent addition of excess cyanide.6 In Table I it is seen that asclepain m is com- pletely inhibited by maleic acid, and is fully reactivated by cyanide. Asclepain s behaves in the same manner (data not recorded).

A plausible explanation of the reversal in these cases is that maleic acid inactivates by oxidizing the enzymes, and that it is itself reduced to the rather stable substance, succinic acid; then cyanide reactivates by reducing the oxidized forms of the enzymes.

6 The same conditions were employed as in measurements with asclepain m and bromelin, except that the hemoglobin method was used to measure proteolytic activity. Following the inactivation by 0.01 M maleic acid, the papain solution (containing 0.6 mg. of enzyme per ml.) was activated to 68 per cent of the full activity value for the cyanide-treated enzyme.

by guest on May 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

770 Plant Proteases. I

The -SH of bromelin apparently does not react readily with maleic acid, as its activity is not appreciably altered by this reagent. When excess cyanide is also added to the solution, the enzyme becomes fully activated.

Heavy Metal Ions-Hellerman (9) explains the inactivation of papain by heavy metal ions as being due to the formation of mercaptides. Activators, such as HCN and HSS, remove the metal and liberate the -SH groups of the enzyme.

When asclepain m and bromclin are inactivated by adding 0.001 N Ag+ or Hg++ to the enzyme solutions, it is possible, by the subsequent addition of excess sulfide or cyanide, to activate asclepain m to 75 per cent, and bromelin to 100 per cent, of the full latent activity. But when 0.01 N metal ions are added, excess sulfide produces 0, or only slight activation, while excess cyanide reactivates asclepain m about 50 per cent, and bromelin about 85 per cent. This depressed reactivation, which results when relatively concentrated solutions of Ag+ or Hg++ are used, is most marked with asclepain s. The activation of this enzyme by 0.001 N Ag+ is completely reversed by 0.1 M H2S (data not recorded). But if 0.01 N Ag+ is used, HZS restores none of the proteolytic activity (3). The reason for this irreversibility is not known.

By use of different concentrations of very dilute silver ion, t,he enzymes can be inactivated to varying degrees. It is interesting to compare the quantities of Ag+ which cause 50 per cent inactiva- tion of a given amount of the different enzyme preparations. These quantities, calculated from the data in Tables I and II, are 2.0 X lo-+ milliequivalent of Ag+ per mg. of asclepain m, and 2.5 X 10d6 milliequivalent of Ag+ per mg. of bromelin. From other published data, the corresponding values for asclepain s (3) and papain (15) were calculated to be 0.8 X lop5 and 1.1 X 1O-5 milliequivalent of Ag+ per mg. of enzyme, respectively.

Xolanain-The results of activation-inhibition experiments with solanain are given in Table IV. It is seen that the behavior of this protease is very different from that of the previous enzymes. Reducing agents, such as cysteine, cyanide, and H2S, have no significant effect on the activity of solanain. Relatively concen- trated solutions of oxidants such as hydrogen peroxide and ferri- cyanide fail to inactivate the enzyme. Iodine in neutral solution

by guest on May 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

D. M. Greenberg and T. Winnick 771

does inactivate solanain, but it appears unlikely that the reaction is one of oxidation. Iodoacetic acid, maleic acid, and cuprous oxide, which react with sulfhydryl compounds, and which com- monly inactivate papainases, have no effect on the activity of solanain. Phenylhydrazine, which activates papain (16) and asclepain s (3), is likewise without effect.

TABLE IV

Activation-Inhibition of Solanain

The untreated enzyme solution, diluted with 2 volumes of water, pro- duced 0.275 to 0.292 milliequivalent of non-protein nitrogen in 6 ml. of digestion mixture.

Reagent added to enzyme solution 1

Inhibitor Activator

None ‘1 ‘I

0.033 M Hz02 0.02 “ K,Fe(CN)s 0.01 “ I* Same 0.03 M iodoacetic acid 0.03 “ maleic acid 5 mg. CUZO 0.02 M phenylhydrazine 0.2 “ NaN02, pH 3.8 Acetate buffer, pH 3.8 0.01 N Ag+ or Hg++ 0.002 “ “ 0.001 “ “ or Hg++ 0.0004 N Ag+ 0.01 N Ag+ or Hg++

0.04 M cysteine 0.1-0.5 M NaCN 0.1 M H,S None

“ ‘I

0.1 M NaCN None

“ I‘ “ “ ‘I “ “ ‘I ‘I

0.1 M H&l or 0.1 M NaCN

Ratio of activity to that of untreated

enzyme

1.6 -1.12 1.10-1.06 1.06 0.96 0.98 0.07 0.13 0.98 0.98 1.00 1.03 0.50 0.94 0.01-0.06 0.14 0.30-0.34 0.56 0.000.13

Philpot and Small (17) have shown that nitrous acid acts on pepsin to produce a yellow diazo compound which has 50 per cent of the original activity. It is interesting to note that HNOz (liberated from NaNOz under comparable conditions) likewise acts on solanain to form a yellow product which has about half the original activity. While the nature of the reaction is not known in the case of solanain, it is possible that phenol groups also are involved as in the case of crystalline pepsin. If this is true, the

by guest on May 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

772 Plant Proteases. I

irreversible inactivation of solanain by iodine could be due to the iodination of phenol groups in the enzyme, similar to the reaction of pepsin (18). Further evidence for this theory is the fact that ketene (at 0” and pH 5.5) produces 50 per cent inactivation in 5 minutes and complete inactivation of solanain within an hour.

Solanain is inactivated by 0.01 N Ag+ or Hg++. As with the previous enzymes, the inactivation by these relatively concen- trated solutions is irreversible. By partial inactivations with more dilute Ag+ solutions, it is found that 1.6 X 10e4 milliequivalent of Ag+ causes 50 per cent inactivation of 1 mg. of solanain. This quantity of Ag+ is about 10 times as great as the corresponding amounts listed for asclepain m, asclepain s, papain, and bromelin. If it is assumed that the impurities in each enzyme have an approx- imately equal effect, the higher value for solanain again suggests that this enzyme has a different active group than the other proteases.

Technical assistance was furnished by the personnel of the Works Progress Administration, Official Project 65-l-08-62, assigned to the University of California.

SUMMARY

1. A study was made of the activation-inhibition reactions of three partly purified plant proteases, bromelin of pineapp!e, asclepain m of the milkweed, Asclepias mexicana, and solanain of the horse-nettle, Solarium elaeagnijolium.

2. The reactions of bromelin and asclepain m resemble those of papain and asclepain s (protease of Asclepias speciosa), and are indicative of the presence of sulfhydryl as a group essential to the activity of these enzymes.

3. Solanain is not affected by oxidizing or reducing agents, or by reagents which react with -SH groups. This enzyme is, therefore, not a papainase.

4. The inactivations produced by nitrous acid and ketene indi- cate that phenolic groups may be essential for the activity of solanain. The course of the inactivation of papain by ketene is

7 It may be of interest that solanain resembles trypsin and chymo- trypsin, but differs from all papainases, in that it does not readily turn casein white during the digestion process.

by guest on May 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

D. M. Greenberg and T. Winnick 773

favorable to the view that phenolic as well as -SH groups are necessary for the activity of this enzyme.

BIBLIOGRAPHY

1. Balls, A. K., and Lineweaver, H., J. Biol. Chem., 130, 669 (1939). 2. Bodansky, A., J. Biol. Chem., 61, 365 (1924). 3. Winnick, T., Davis, A. R., and Greenberg, D. M., J. Gen. Physiol.,

23, 275, 289, 301 (1940). 4. Northrop, J. H., and Kunitz, M., J. Gen. Physiol., 16, 313 (1932). 5. Anson, M. L., J. Gen. Physiol., 22, 79 (1938). 6. Anson, M. L., J. Gen. Physiol., 23, 321 (1940). 7. Hellerman, L., in Cold Spring Harbor symposia on quantitative bi-

ology, Cold Spring Harbor, 7, 165 (1939). 8. Balls, A. K., and Lineweaver, H., Nature, 144, 513 (1939). 9. Kellerman, L., Physiol. Rev., 17, 454 (1937).

10. Hellerman, L., and Perkins, M. E., J. BioZ. Chem., 107, 241 (1934). 11. Fruton, J. S., and Bergmann, M., J. BioZ. Chem., 133, 153 (1940). 12. Herriott, R. M., and Northrop, J. H., J. Gen. Physiol., 18, 35 (1934).

Herriott, R. M., J. Gen. Physiol., 19, 283 (1935). 13. Maschmann, E., Biochem. Z., 279, 225 (1935). 14. Morgan, E. J., and Friedmann, E., Biochem. J., 32, 862 (1938). 15. Krebs, H. A., Biochem. Z., 220, 289 (1930). 16. Balls, A. K., and Hoover, S. R., J. BioZ. Chem., 121,737 (1937). 17. Philpot, J. St. L., and Small, P. A., Biochem. J., 32, 542 (1938). 18. Herriott, R. M., J. Gen. Physiol., 20, 335 (1937). by guest on M

ay 28, 2020http://w

ww

.jbc.org/D

ownloaded from

David M. Greenberg and Theodore WinnickREACTIONSACTIVATION-INHIBITION

PLANT PROTEASES: I.

1940, 135:761-773.J. Biol. Chem. 

  http://www.jbc.org/content/135/2/761.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  tml#ref-list-1

http://www.jbc.org/content/135/2/761.citation.full.haccessed free atThis article cites 0 references, 0 of which can be

by guest on May 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from