THE LEUCYLPEPTIDASES OF MALT, CABBAGE, AND SPINACH*

BY JULIUS BERGER AND MARVIN J. JOHNSON

(From the Departments of Biochemistry and Agricultura.! Bacteriology, University of Wisconsin, Madison)

(Received for publication, July 24, 1939)

In 1929, Linderstrom-Lang and Sato (1) and Linderstrom- Lang (2) suggested that one enzyme in green malt and hog erepsin appeared to be able to hydrolyze both leucylglycine and leucyldi- glycine at equal rates, and in 1936, Johnson et al. (3) also showed that purified leucylpeptidase of hog erepsin hydrolyzed leucyl- glycine and leucyldiglycine at equal rates at pH 8. The hydroly- sis of these peptides was activated by magnesium ions, a property which appeared to be an outstanding characteristic of leucyl- peptidase. Recent work (4) has shown that Mn ions also ac- tivate hog leucylpeptidase. It therefore appeared desirable to reinvestigate the peptidases of malt and other plants, with special regard to their metal activation.

By a simple purification involving only one acetone precipita- tion at slightly acid reaction, it has been found possible to make from malt, cabbage, and spinach extracts leucylpeptidase prepa- rations which are practically identical in specificity and metal activatability with purified hog leucylpeptidase. Often, how- ever, small amounts of dipeptidases and still smaller amounts of a different polypeptidase survive the acetone precipitation. The complete recovery of leucylglycine- and leucyldiglycine-splitting activity in the acetone-precipitated enzyme preparations from two of the three plants would suggest that in the original crude

* Supported in part by a grant from the Wisconsin Alumni Research Foundation.

Published with the approval of the Director of the Wisconsin Agri- cultural Experiment Station.

655

by guest on March 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

656 Plant Leucylpeptidases

extracts leucylpeptidase was mainly responsible for the hydrolysis of leucylglycine and leucyldiglycine.

In addition to a leucylpeptidase activated by both magnesium and manganese ions, all three plants studied contained enzymes whose diglycine but not alanylglycine splitting was activated by Mn ions. Contrary to previous literature (5), crude malt ex- tracts do contain peptidases capable of hydrolyzing d-leucyl- glycine, although the racemic mixture is hydrolyzed approximately 50 times as rapidly.

EXPERIMENTAL

Methods

Cabbage extract.s were made by grinding fresh, green leaves of new cabbage in a food chopper and pressing out the juice through cheese-cloth. This extract was then clarified by filtration with Hyflo super-cell and used as the source of cabbage peptidases.

Malt enzyme preparations were made from freshly malted green barley malt which was dried at room temperature and then finely ground. This ground malt was extracted at room tempera- ture for 3 to 4 hours at the natural pH of the suspension (usually pH 5.8 to 6.0), with 5 times its weight of distilled water. The extract was clarified by filtration with Hyflo super-eel. Ground, dried malt stored for 1 year at room temperature was found to be just as active as freshly dried malt. If fresh, moist malt was ground and exbract.ed as above, only 25 per cent as much leu- cylpeptidase activit)y was obtained.

Spinach enzymes were prepared from fresh spinach leaves in a manner similar to that for preparation of the cabbage extract. However, the clarified spinach extracts had to be dialyzed before analysis in order to remove substances which interfered with the reading of the end-point in the peptidase determination.

The methods of enzyme determination were the same as those used in previous work (4). Racemic substrates were always pres- ent in ~/15 concentration, and others in M/~O concentration. The pH was 8.0 f 0.2 in all cases. In the case of dl mixtures, per cent hydrolysis of one linkage of one component is indicated.

1 A filter aid sold by Johns-Manville.

by guest on March 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

J. Berger and M. J. Johnson

Results

657

In Table I it may be seen that crude malt extract hydrolyzed leucyl peptides much more rapidly than alanyl or glycyl peptides, with the exception of alanylglycine. After precipitation with acetone, all of the leucyl peptide-hydrolyzing activity was re-

TABLE I

Peptidases of Malt

Preparation I

Crude extractt

Substrate -

l- Time

of incu-

bath

I

hrs.

dl-Alanylglycine.. 0.5 dl-Leucylglycine. . . 0.5 Diglycine . . . . . 9.7 dl-Alanyldiglycine 9.7 dl-Leucyldiglycine . 0.5 Triglycine. . . . . 9.7

d

L

. _

Pe+ cent 70 52 44 76 40 28

- e

_

t

-

ketone-precipitatec -Xl5yDFJ$

t Hydrolysis riz L

incu- Con- mtion trol

~-

Am. w cent

0.5 48 0.5 64

20 56 20 60 0.5 68

20 16

-

~- Per

cent

48 84 58 70 84 16

Per cent

53 92 47 30

127 22

e ! lz

-.

Preparation II*

Ihe of

incu- hior

hrs.

1 2.5 6 6 2.5 6

-

‘(

-

Hydrolysis

:on- 0.01 M trol MgClr

~-

Per Per cent cent 39 38 26 56 32 20 36 38 22 58 10 9

* 450 cc. of crude enzyme solution were precipitated with 1 volume of acetone, and the precipitate was suspended in 125 cc. of water. 0.10 cc. of the clarified solution was used in 3 cc. of reaction mixture for leucylglycine and leucyldiglycine analyses, while 1.0 cc. was used for the other four peptides.

t 1 cc. of enzyme solution (representing 0.2 gm. of dry malt) was used in 3 cc. of reaction mixture.

$20 cc. of crude enzyme solution at pH 5.8 were mixed with 20 cc. of acetone, and the resulting precipitate centrifuged and suspended in 15 cc. of distilled water, adjusted to pH 7.0, and recentrifuged. 1.0 cc. of clear solution was used in 3 cc. of reaction mixture.

8 Based on activities in the absence of added metals.

covered, but only 20 to 50 per cent of the alanyl and glycyl pep- tide-splitting activities. The leucyl peptides were hydrolyzed at equal rates, as Linderstrom-Lang and Sato (1) had found, and this hydrolysis was activated by Mg or Mn (Table II) ions. It has been shown in a previous paper (4) that, of a large number of

by guest on March 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

658 Plant Leucylpeptidases

ions tested, only Mg and Mn are capable of activating malt leucylpeptidase.

TABLE II

Metal-Activated Malt Peptidases

EtOH-acetone preparation’

Substrate Hydrolysis -

; . _

Pm cent

dl-Alanylglycine.. 19 dl-Leucylglycine.. . 22 Diglycine.. . . . . . . . . . . . . 8 dl-Alanyldiglycine.. . 5 dl-Leucyldiglycine.. . . 16 Triglycine.. . . . . . . . . . 4

- 0.01 M !d’gso4 il

Pm cent

22 36 9 4

25 2

‘Loo1 Y : il InSO I

Per cent 22 48 13 16 34

8 -

Acetone-precipitated Preparation IVt

The of

ncuba- tion

.-

Hydrolysis

hrs. Pm P@ per cent cent cent

3 38 38 34 3 26 36 48 3 10 9 34 9 31 23 50 4 28 34 46

24 13 13 17 I

* 67 cc. of acetone-precipitated Preparation II at pH 5.9 were mixed with 67 cc. of EtOH and centrifuged after 3 minutes. The precipitate was suspended in 28 cc. of Hz0 and recentrifuged. 1.0 cc. of the resulting clear solution was used in 3 cc. of reaction mixture, except for leucylglycine and leucyldiglycine (0.5 cc.). The incubation time was 14 hours.

t This preparation was made similarly to Preparation II in Table I, but it was allowed to age in the refrigerator at 2” for 10 days. 1.0 cc. of enzyme solution was used in 3 cc. of reaction mixture, except for leucyl- glycine and leucyldiglycine (0.3 cc.).

.ool Y fnSO4

TABLE III

Effect of Mg and Mn Concentration on Peptide Hydrolysis by Malt Leucylpeptidase

0.26 cc. of acetone-precipitated enzyme preparation was used in 3 cc. of reaction mixture. The incubation time was 1 hour.

I Per cent hydrolysis of one linkage of one component

Substrate

%i MgClt 0.02 M MnSO4

0.005 ?d 0.01 Y 0.03 Y 0.05 Y NaC1 k-4 M 10-r Y 10-t Y ------- --

dl-Leucylglyoine.. . 21 36 44 45 41 18 39 46 37 dl-Leucyldiglycine.. . . 18 36 38 40 40 18 42 48 45

In Table II, data obtained with two different preparations show that both Mg and Mn ions accelerated leucylglycine and leucyl-

by guest on March 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

J. Berger and M. J. Johnson 659

diglycine hydrolysis, but only Mn ions accelerated diglycine, alanyldiglycine, and triglycine hydrolysis. These facts might be interpreted to indicate that two polypeptidases are present in the preparations, but recent work (4) has shown the possibilities of anomalous behavior in the metal activation of peptidases.

The effect of Mg and Mn concentration on the rate of hydroly- sis of leucylglycine and leucyldiglycine by malt leucylpeptidase is shown in Table III. With magnesium, the amount of activation

FIG. 1. pH optima for hydrolysis of leucylglycine and leucyldiglycine (Preparation II) by an acetone-precipitated leucylpeptidase preparation from malt, in the presence of 0.001 M MnS04, and of leucyldiglycine (Prep- aration I) by a crude malt extract (dialyzed 4 days) in the presence of 0.003 M MgClz. The incubation time was 1 hour at 40” in all cases. Equal amounts of enzyme were used in all determinations with leucylglycine and leucyldiglycine (Preparation II).

increased until a concentration of 0.03 to 0.05 M was reached, while 0.001 M Mn was optimal. 0.02 M NaCl did not cause ac- tivation, indicating that the cation of MgClz was responsible for activation.

From Fig. 1, it may be seen that in the presence of 0.001 M

Mn an acetone-precipitated malt enzyme preparation hydrolyzed leucylglycine most rapidly at pH 9.1 to 9.5, and leucyldiglycine at pH 8.3 to 8.9. In the presence of 0.003 M MgC12, leucyldi-

by guest on March 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Plant Leucylpeptidases

glycine was hydrolyzed most rapidly at pH 8.8 by a dialyzed, crude malt extract. For pH-activity determinations in these and other experiments, substrates half neutralized with NaOH were adjusted to the desired pH by the addition of predetermined amounts of 1 N acetic acid or 1 N NaOH solution. Thus there was no buffering other than that provided by the peptides themselves in the neighborhood of pH 8 and by the small amount of acetate at the lower pH values. Linderstrom-Lang and Sato (1) reported optimum rates of hydrolysis of leucylglycine and leucyldiglycine by malt peptidases at pH 8.6, but this was in the absence of any metal activator.

When a malt enzyme hydrolysis of dkleucylglycine at pH 8 in the presence of 0.001 M Mn ions was allowed to incubate with toluene at 40” for 24 hours, it was found that a greater hydrolysis (136 per cent) was obtained than could be accounted for if only one optical isomer were split. Confirmatory experiments were therefore made with crude malt extract which was concentrated in vucuo below 30” to one-tenth its original volume and then di- alyzed. This procedure was repeated, so that the final enzyme solution was 5.5 times as concentrated as the original. d-leucyl- glycine and d-leucyldiglycine obtained from Hoffmann-La Roche, Inc., were then incubated with this enzyme solution. The re- sults are shown in Table IV, which also contains hydrolysis values obtained with the same enzyme solution on diglycine, dl-leucyl- glycine, and dl-leucyldiglycine. It may be seen that d-leucyl- glycine was hydrolyzed to the extent of 75 per cent in 17 hours, or at approximately one&tieth the rate at which dl-leucylglycine was split. However, the hydrolysis of d-leucylglycine was ac- tivated by Mn but not Mg ions, whereas dl-leucylglycine hydroly- sis was activated by both Mg and Mn. Furthermore, acetone precipitation destroyed most or all of the d-leucylglycine activity without appreciably affecting the dl-leucylglycine activity. It is therefore likely that leucylpeptidase, which is responsible for most of the I-leucylglycine hydrolysis, does not split d-leucylgly- tine and that another peptidase, possibly an Mn-activated di- glycine-splitting dipeptidase, is responsible. d-Leucyldiglycine incubated with the concentrated crude enzyme in the presence and absence of 0.01 M MgSOl and 0.001 M MnSOl gave hydrolysis values within the limits of experimental error, so that it is still uncertain whether it is hydrolyzed at all.

by guest on March 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

J. Berger and M. J. Johnson

Tables V and VI present the data obtained on the distribution and metal activation of the peptidases in crude cabbage extracts

TABLE IV

Hydrolysis of cl-Leucylglycine by Malt Peptidases

Hydrolysist

Substrate* Time of

incubation Control 0.01 ?d 0.001 M

M&O4 M&O4 ~____

bra. pet cent per cent per cent

d-Leucylglycine.. 17 18 18 75 dl-Leucylglycine. 1 19 39 45

d-Leucyldiglycine.. 17 2 1 4

dl-Leucyldiglycine.. 1 19 28 42 Diglycine. 1 18 20 50

* d-Leucylglycine and cl-leucyldiglycine were present in M/30 con-

centration, pH 8. t These values have been corrected for the enzyme control. With

d-leucylglycine, d-leucyldiglycine, and diglycine, 1.0 cc. of concentrated enzyme solution, and with dl-leucylglycine and dl-leucyldiglycine, 0.25 cc.

of this enzyme solution were used in 3 cc. of reaction mixture.

TABLE V

Peptidases of Cabbage (Preparation I)

I Crude enzyme* Acetone-precipitated ensymet

Substrate

dl-Alanylglycine.

dl-Leucylglycine. Diglycine dl-Alanyldiglycine..

dl-Leucyldiglycine.. Triglycine.

Time of incuba-

tion

hrs. per cent hru. pm cent per cent per cent

7 36 2 2 4 10 2 44 1 14 40 91 7 16 7 2 4 6 7 50 1 -2 1 4 2 48 1 16 50 104

7 16 7 2 2 3

Hydrolysis

Control

1;gFY

recipita- tion

* 0.5 cc. of crude enzyme extract was used in 3 cc. of reaction mixture.

t 20 cc. of crude extract diluted with 20 cc. of water (pH 6.26) were precipitated with 40 cc. of acetone and centrifuged after 1 minute. The precipitate was suspended in 10 cc. of distilled water (pH 7.4) and centri-

fuged. 1.0 cc. of the resulting clear solution was used in 3 cc. of reaction mixture, except for leucylglycine and leucyldiglycine (0.5 cc.).

and in acetone-precipitated solutions. Crude extracts hydro- lyzed leucyl peptides much more rapidly than alanyl or glycyl

by guest on March 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

662 Plant Leucylpeptidases

peptides, and acetone precipitation destroyed or removed most of the small amount of alanyl and glycyl peptide-splitting enzymes originally present. It may be seen that cabbage contains a leucyl- peptidase which hydrolyzes leucylglycine and leucyldiglycine at equal rates and that this hydrolysis is activated by both Mg and Mn ions. In Table VI, detailed analysis of the acetone-precipi- tated enzyme solution was made 17 hours after the precipitation was carried out, and during this time the enzyme preparation had been kept in the refrigerator at 2’. Whereas it appears that only 40 per cent of the leucylglycine- and leucyldiglycine-

TABLE VI

Peptidases of Cabbage (Preparation II)

Crude enzyme*

Substrate T&e 1 Hydrolysis of ~

incu- COW bation trol --

hrs. per Cenl

dl-Alanylglycine.. . . 2 32 dl-Leucylglycine. 1 9 Diglycine.. . 2 8 dl-Alanyldiglycine.. 2 20 dl-Leucyldiglycine.. . 1 12 Triglycine.. . . . . . . 2 8

Tl

IBT ceni

30 34 14 24 36

6

- 1

6

1

-

Acetone-precipitated enaymet R4

‘I Hydrolysis I OOVWY after

‘2 ncu- atiol 1

hr.% P

2 1 2 2 1 4

-

0

- ! ps

precipi- ‘401 M 0.01 rd tation Mn Mg --- er cent per cent pm cent 40 27 56 23 33 24 8 43 30 6 31 54 37 8 12

* 0.5 cc. of crude extract was used in 3 cc. of reaction mixture. 7 125 cc. of crude extract at pH 6.35 were precipitated with 125 cc. of

acetone and centrifuged after 5 minutes. The precipitate was suspended in 50 cc. of distilled water and centrifuged. 1.0 cc. of the resulting clear solution was used in 3 cc. of reaction mixture.

hydrolyzing activity was recovered, analyses made on the same solution within 1 hour of its preparation actually showed 108 per cent recovery. Complete recovery of leucylpeptidase was also obtained in Preparation I. The instability of acetone-precipi- tated preparations at pH 7 and 2” has frequently been noted, but usually there has been a more gradual loss in activity. For ex- ample, after 1 week’s storage at 2’, malt leucylpeptidase Prepara- tion IV decreased to one-third of its original activity; with another preparation, one-third of the original activity remained after 5 weeks at 2’.

by guest on March 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

J. Berger and M. J. Johnson 663

In Fig. 2, it may be seen that cabbage leucylpeptidase in the presence of 0.003 M MgC12 hydrolyzes leucylglycine at a pH op- timum of 8.9 and leucyldiglycine most rapidly at pH 8.2 to 9.0.

Table VII summarizes the data obtained on spinach peptidases. Here again the acetone-precipitated solution hydrolyzed leucyl- glycine and leucyldiglycine at approximately equal rates, and both Mg and Mn ions activated these hydrolyses. About 50 per cent of leucylglycine- and leucyldiglycine-hydrolyzing activity was

I PER CENT HYDROLYSIS

I+ 8 9 IO

FIG. 2. pH optima for hydrolysis of leucylglycine and leucyldiglycine by an acetone-precipitated leucylpeptidase preparation from cabbage, in the presence of 0.003 M MgCla. The incubation time was 1 hour. Twice the amount of enzyme was used for leucylglycine as for leucyldiglycine determinations.

recovered after acetone precipitation. 0.01 M NaCl had no ac- tivating effect.

The curves of Fig. 3 show that in the presence of 0.001 M

MnS04, spinach leucylpeptidase hydrolyzed leucylglycine and leucyldiglycine most rapidly at pH values of 9.2 and 8.6, respec- tively. With hog leucylpeptidase (3), hydrolysis was never ob- tained at pH values at which only inappreciable amounts of pep- tide anion were present; all three plant leucylpeptidases behaved similarly; below pH 7 very little hydrolysis occurred.

by guest on March 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

664 Plant Leucylpeptidases

After the similarity of the plant and animal leucylpeptidases with respect to metal activatability was demonstrated, the ques- tion arose as to the similarity of their specificity for the hydrolysis of various peptides. In Table VIII, the specificities of malt, cabbage, and hog leucylpeptidases are compared. In most in- stances, the peptides and their substituted derivatives were hy- drolyzed by the various preparations at practically the same rates. The outstanding difference was the inability of the plant enzymes

TABLE VII

Peptidases of Spinach

Crude extract*

Substrate

dl-Alanylglycine . . dl-Leucylglycine. . Diglycine. dl-Alanyldiglycine dl-Leucyldiglycine.. Triglycine . .

---- hrs. per cent per cent pet ceni

1 26 24 16 1 23 31 25 2$ 9 5 1 24 16 14 1 24 28 28 1 15 14 6

1 .

Acetone-precipitated enzymet

Hydrolysis

__--- hrs. per cent per cent pet cent

3 38 40 20 2 20 56 59 3 3 1 22 3 5 5 6 2 23 45 48 3 2 1 1

* Crude spinach juice was dialyzed against cold running tap water for 24 hours; 1.0 cc. of enzyme solution was used in 3 cc. of reaction mixture.

t The above preparation was dialyzed 40 hours’against distilled water in a refrigerator; then it was precipitated at pH 6.07, with an equal volume of acetone. The precipitate from 55 cc. of enzyme solution was suspended in 20 cc. of water and recentrifuged. 1.0 cc. of clear extract was used in 3 cc. of reaction mixture.

$ After 15 hours incubation, diglycine hydrolysis was 40 per cent in the control, and 82 per cent in the presence of 0.001 M MnSOd.

(especially malt) to hydrolyze glycyl-dl-leucylglycine as rapidly as did hog leucylpeptidase. Separate determinations were made on the hydrolysis of 0.05 M dl-leucyldiglycine and 0.05 M glycyl- dl-leucylglycine in the presence of 0.003 M MgC12. Hog leucyl- peptidase hydrolyzed these peptides at approximately equal rates, if the comparison was made before extensive hydrolysis occurred of the dl-leucylglycine set free from the glycyl-dl-leucylgiycine. Cabbage leucylpeptidase, similarly analyzed, split dl-leucyldigly-

by guest on March 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

J. Berger and M. J. Johnson 665

tine 1.7 times as rapidly as glycyl-&leucylglycine, whereas malt leucylpeptidase split dl-leucyldiglycine 4 times as fast. Alanyl- diglycine was split at a much slower rate by malt and cabbage than by hog leucylpeptidase. Leucylmethylamine was not split at all by malt or cabbage, whereas the hog enzyme did hydrolyze this decarboxylated peptide very slowly. These three differences were confirmed when a hog leucylpeptidase other than the one reported by Johnson et al. (3) was analyzed. The rapid hy-

PH FIG. 3. pH optima for hydrolysis of leucylglycine and leucyldiglycine by

an acetone-precipitated leucylpeptidase preparation from spinach, in the presence of 0.001 M MnS04. The incubation time was 1 hour. Equal

amounts of enzyme were used in all determinations.

drolysis of alanylglycine by the cabbage preparation in Table VIII was undoubtedly due to some contaminating dipeptidase, since other preparations from cabbage (Tables V and VI) have shown much slower hydrolysis of this peptide. Furthermore, the hydrolysis of alanylglycine by this preparation was inhibited by Mg and Mn ions.

The possibility exists, of course, that these differences in spec- ificity are caused by accompanying substances that influence differently the action of the same enzyme on different substrates. However, while this factor is possibly of some influence, it seems

by guest on March 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

666 Plant Leucylpeptidases

very likely that leucylpeptidases from two sources as widely dif- ferent as hogs and plants would exhibit some differences in spec- ificity. In fact, it is remarkable that leucylpeptidases from

TABLE VIII

Specificities of Hog, Malt, and Cabbage Leucylpeptidases -

- Substrata Per writ hydrolysis of one linkage in

dl-Leucyldiglycine. ......... dl-N-Methylleucyldiglycine Glycyl-dl-leucylglycinei .... dl-Alanyldiglycine. ......... Triglycine. ................. Sarcosyldiglycine. .......... Tetraglycine. ............... dl-Leucylglycine. ........... dl-Leucylmethylamine. ..... Diglycine. .................. Glycylmethylamine ......... dl-N-Methylleucylglycine*. dl-Alanylglycine* ........... dl-Prolylglycine*. .......... dl-Prolyldiglycine*. ......... dl-Leucyldiglycine -I- 0.001 M

MnSOJ*. .................

- 1

-- hr.

39 0

76t 10 0 0 0

40 6 1 0 0

5 24 lr*. lm. --

101 110 0 5

194 206 53 112 3 12 0 2 2 6

100 105 10 17 2 13 0 -1 4 21

0 5 24 -1 4 23

99 113 146 -

ht. 1 1 _- - .-

30 1 8 4 1 2 0

34

1 0 0 0 0 2

34 -

a Ire. - 84 0

40 8 2 4 1

96 0 6 1 3

18 2 4

80 -

24 .X8. 1 hr. --

110 27 2 0

88 20 22 3 4 1 6 -1 2 0

LO6 32 2 0

14 4 0 1 5 0

60 41 8 4 8 3

121 58 __- -

A. lz:. -- 83 112 4 8

88 160 12 36 2 10 0 4 3 7

100 110 2 2 8 14 2 4 2 6

86 92 16 40 6 16

98 126

* The data on hog leucylpeptidase are those from which Table IV of the paper by Johnson et al. (3) was made, with the exception of the last five peptides; here, a different preparation, which gave 76 per cent hydrolysis of dl-leucyldiglycine in 1 hour, was used.

t The sparingly soluble glycyl-dl-leucylglycine was present in ~/30 concentration. M/300 MgClt was present in all cases, except the last. The enzyme concentration was held constant.

1 A check with original data indicated that the figure published previ- ously (3) was in error, and should have been 76 per cent instead of 38 per cent.

widely varying sources should be so similar in activation, spec- ificity, and pH optima.

In a previous paper (4) it was reported that crude malt, spinach, and cabbage extracts contained peptidases whose hydrolysis of

by guest on March 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

J. Berger and M. J. Johnson

diglycine, but not alanylglycine, was activated by Mn ions. In the case of malt (the others were not tested) Co++ ions also activated diglycine hydrolysis. Mn activation of diglycine splitting has also been noted with acetone-precipitated prepara- tions from malt (Table II), from cabbage (Table VI), and from spinach (Table VII).

SUMMARY

1. The peptidase systems of three plants have been investigated by the study of the hydrolysis of six peptides by crude extracts and by purified preparations.

2. Malt, cabbage, and spinach contain a leucylpeptidase which hydrolyzes the peptides leucylglycine and leucyldiglycine at approx- imately equal rates; this hydrolysis is activated by Mg or Mn ions.

3. The peptidases of crude malt extracts hydrolyze d-leucyl- glycine but at a much slower rate than dl-leucylglycine.

4. Malt and cabbage leucylpeptidases are very similar in specificity to hog leucylpeptidase; the outstanding observed dif- ference is their inability to hydrolyze glycyl-dl-leucylglycine and alanyldiglycine as rapidly as does hog leucylpeptidase.

BIBLIOGRAPHY

1. Linderstr$m-Lang, K., and Sato, M., Z. physiol. Chem., 184, 83 (1929).

2. Linderstr$m-Lang, K., Z. physiol. Chem., 182,151 (1929); 188,48 (1929). 3. Johnson, M. J., Johnson, G. H., and Peterson, W. H., J. Biol. Chem.,

116, 515 (1936). 4. Berger, J., and Johnson, M. J., J. Biol. Chem., 130,641 (1939). 5. Liiers, H., and Malsch, L., Woch. Brau., 46,265 (1929).

by guest on March 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Julius Berger and Marvin J. JohnsonCABBAGE, AND SPINACH

THE LEUCYLPEPTIDASES OF MALT,

1939, 130:655-667.J. Biol. Chem. 

  http://www.jbc.org/content/130/2/655.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/130/2/655.citation.full.haccessed free atThis article cites 0 references, 0 of which can be

by guest on March 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from