THE EFFECT OF ZINC ON ALKALINE PHOSPHATASES” · THE EFFECT OF ZINC ON ALKALINE PHOSPHATASES” BY...

THE EFFECT OF ZINC ON ALKALINE PHOSPHATASES”

BY E. HOVE, C. A. ELVEHJEM, AND E. B. HART

(From the Department of Biochemistry, College of Agriculture, University of Wisconsin, Madison)

(Received for publication, March 27, 1940)

Zinc has been shown to be an element essential to the life and growth of rats, but as yet there is no clear indication of what its function may be in metabolism. There are several reports of the occurrence of zinc in pure biological compounds. Scott and Fisher (1) have shown zinc to be necessary for the crystallization of insulin, although other metals were also effective. Keilin and Mann (2) report that carbonic anhydrase contains 0.32 per cent zinc. However, it has been well established that this enzyme is inhibited by small amounts of added zinc (Meldrum and Roughton (3), and unpublished data from this laboratory). In connection with the role of this red blood cell enzyme it may be significant that a zinc-porphyrin compound is excreted in the urine of hu- mans (4). A zinc-porphyrin has also been isolated from diptherial toxic broth (5). Foster and Waksman (6) have shown zinc to be essential for the growth and fumaric acid production of Rhizopus nigricans. Recently Holmberg (7) has found that a purified uricase preparation contains 0.13 per cent zinc.

In previous work from this laboratory (8, 9) we have been able to find no detectable difference in the carbohydrate metabolism of zinc-deficient rats. However, a disturbance of the nitrogen metabolism was observed. This involved a decreased blood non- protein nitrogen, a marked delay in the rate of absorption of amino acids fed by mouth, an increase in fecal nitrogen excretion, a decrease in the daily urinary creatinine excretion, and a marked

* Published with the approval of the Director of the Wisconsin Agricul- tural Experiment Station.

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

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426 Effect of Zinc on Phosphatases

reduction in the pancreatic trypsin content. It was also shown that there is no appreciable decrease in the zinc content of most of the soft tissues of the zinc-deficient animal. However, bone, teeth, and to a lesser degree small intestines do show a decreased concentration of this element. Because of this change our at- tention was attracted to the possible effect of zinc ions on the activity of phosphatases of the bone and other tissues.

A survey of the literature reveals that Erdtman (10) found a 0.004 mM concentration of zinc ions to produce an 18 per cent inhibition of dialyzed kidney phosphatase. Jenner and Kay (11) state that zinc has no, or slight inhibitory, effect on phosphatases, although they give no experimental details or data. However, Pett and Wynne (12) observed a stimulation of bacterial pyro- phosphatases by zinc in small amounts and inhibition at higher levels. Other bacterial phosphatases were inhibited by all concentrations of zinc used.

EXPERIMENTAL

Bone phosphatase preparations were made by grinding the cleaned fresh bone (rat femurs), making up to a 5 per cent suspension in water, and allowing to autolyze for 24 hours at 37” in the presence of chloroform. This mixture was then filtered through a Whatman No. 42 paper, producing a water-clear enzyme preparation containing about 4 mg. of dry matter per cc. The crude intestinal enzyme was prepared in the same way ex- cept that the washed, whole, rat intestine was minced and made up to a 10 per cent suspension. After the filtration this crude intestinal phosphatase solution had about 14 mg. of dry matter per cc. When this was dialyzed at pH 6.1 for 48 hours, the dry matter content was reduced to 2 mg. per cc. The enzyme solution took on a non-filtrable cloudiness during dialysis which precipitated after a few days in the refrigerator. Toluene was used as a preservative during the dialysis, and a mixture of toluene and chloroform afterwards.

The substrate used in a majority of the experiments to be reported has been sodium fl-glycerophosphate, although disodium phenyl phosphate, sodium hexosediphosphate, and sodium pyrophosphate have also been used. These phosphate esters were made up as a 4.8 mM solution (1 cc. = 0.15 mg. of P) in a 50


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Hove, Elvehjem, and Hart 427

mM Verona1 buffer. The final pH was adjusted to 9.2. For each determination 10 cc. of the substrate solution and 0.5 cc. of the enzyme solution in a total volume of 11.5 cc. were incubated at 37”. The reaction was stopped with 2 cc. of a 10 per cent trichloroacetic acid solution, filtered if necessary, and the liberated inorganic phosphorus determined by the customary Fiske-Sub- barow method. Blanks were run with every experiment. Any substance added to the reaction mixture was tested for its effect on the substrate alone, under the identical time and temperature conditions of the reaction.

Effect of Zinc on Phosphatases in Vitro-In the preliminary experiments on the effect of zinc on the activity of animal alkaline phosphatases in vitro the standard King-Armstrong method was used. The substrate for this method is disodium phenyl phosphate, and the incubation time is half an hour at 37.5”. It was found that the activity of crude bone phosphatase was progressively inhibited by increasing concentrations of zinc ion. The effect of zinc on the action of crude kidney phosphatase was also inhibitory, but only to a small degree. However, when crude intestinal phosphatase was tested, zinc was found to have no effect at all.

These preliminary results appeared to indicate a difference in the phosphatases from the three sources, so the work was extended to other substrates. The effect of zinc on the hydrolysis in vitro of fl-glycerophosphate (21 hours incubation) by the crude bone and kidney phosphatases was more or less markedly inhibitory, thus agreeing with the first results obtained with the phenyl phosphate substrate. However, the effect of zinc on the hydrolysis of glyccrophosphate by the crude intestinal phosphatase was markedly activating. Zinc also was found to activate the hydrolysis of sodium hexosediphosphate and sodium pyrophosphate by the crude intestinal phosphatase. These results are shown in Fig. 1. During the last 12 months this stimulating effect of zinc on the hydrolysis of /?-glycerophosphate by crude rat intestinal phosphatase has been repeated on approximately 60 to 70 indi- vidual preparations, the degree of activation varying between 40 and 110 per cent. Zinc chloride, sulfate, and nitrate are equally effective. The pH of the reaction mixture is not altered by the amounts of zinc salt used in these experiments.


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The time course of the hydrolysis of P-glycerophosphate by the crude intestinal phosphatase alone, and with optimum zinc, optimum magnesium, and a combination of optimum amounts of zinc and magnesium, is shown in Fig. 2. From the curves in this chart it can be seen that both of the metal ions undergo an aug- mentation in their percentage activation effect with time. This has previously been observed for the magnesium ion by Bodansky (13) and Holmberg (14). It should be noticed that the percent-

mb! Zinc FIG. 1. The effect of zinc on crude intestinal phosphatase activity in

vitro. 21 hour incubation period at 37”, pH 9.2. Substrates, Curve I, hexosediphosphate; Curve II, glycerophosphate; Curve III, sodium pyrophosphate.

age activation due to zinc at 3 hour’s incubation is slight, amount- ing to only 5 per cent. This furnishes a possible explanation of the lack of zinc activation of the hydrolysis of the phenyl phosphate substrate by the crude intestinal phosphatase, since here the incubation was only continued for a half hour; and in fact, it has been found that with a longer incubation period zinc does activate the hydrolysis of this substrate. Since the metal ions, zinc and magnesium, together produce an effect greater than


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either alone (Fig. 2), it seems possible that the mechanism of activation by these ions is, in part at least, different and independent.

Dialysis of the crude intestinal phosphatase resulted in a complete loss of the ability of zinc ions to activate the enzyme. In fact the intestinal enzyme after dialysis was inhibited by zinc; this observation brings a uniformity into the action of zinc ions on phosphatases from the three sources-bone, intestine, and kid-

54ot /r/ - ont rol -I

VI 1111 I I I I 4 6 d 10 14

1 2 12 16 18

Time (hours ) FIG. 2. The time course of the hydrolysis of fl-glycerophosphate by

crude intestinal phosphatase. Concentration of zinc, 0.03 mM; of magnesium, 0.1 mM.

ney. The degree of the zinc inhibition of intestinal phosphatase increases slowly with the length of dialysis, as is shown in Fig. 3. From the curves shown in Fig. 3, it is evident that the ability of zinc to stimulate the magnesium-activated dialyzing enzyme outlasts the ability of zinc to stimulate the dialyzing enzyme alone. However, this property also is lost when dialysis is suffi- ciently complete, as is shown in Preparations 2 and 3 of Table I. Also from Table I it is evident that the dialysate, when again added to the dialyzed intestinal enzyme, can restore the zinc


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activatability of the enzyme. The dialysate alone, or in the presence of zinc or magnesium or both, had absolutely no effect on the substrate.

These results indicate that there is present in crude intestinal preparations, but seemingly not in bone or kidney preparations, a dialyzable substance which, together with zinc ion, is able to activate phosphatases. This dialyzable zinc coactivator is not destroyed by taking the dialysate to dryness at 100”. It is de-

FIG. 3. The change of metal ion action on intestinal phosphatase activity with increasing time of dialysis. Substrate, P-glycerophosphate; incubation period, 21 hours; concentration of zinc, 0.03 mM; of magnesium, 0.2 mM.

stroyed by ashing. It is soluble in water, partially soluble in absolute ethyl alcohol, and completely insoluble in pyridine, acetone, and absolute butyl alcohol. It is soluble in dilute acids and bases, although it seems to be partially destroyed by being taken to dryness in the presence of concentrated ammonia or NaOH. A boiled tissue extract from a fresh intestine has none of this substance. A boiled tissue extract from a portion of the same intestine after a 24 hour autolysis at 37” has high activity.

Since the zinc coactivator appeared to be a product of autolysis,


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a casein hydrolysate was tried and found to have a high activity, as is shown in Table II. Following this observation, a group of pure amino acids was tested (the pH of the amino acid solutions having been adjusted before use). As is evident from Table II, all of the a-amino acids had a more or less strong zinc-coactivating property. Their effect seemed to a degree to be inversely related to the length of the chain. The corresponding p-amino acids, keto acids, hydroxy acids, or aliphatic acids had little or no ac-

TABLE I

E$ect of Dialysis for S6 Hours and of Metal Ions on Intestinal Phosphatase Preparations

Substrate, P-glycerophosphate; incubation period, 21 hours at 37”; final concentration of added metal ions, Zn 0.05 mM, Mg 0.2 mM.

None

Mg Zn Mg + Zn None

Mg Zn Mg + Zn None

Mg Zn Mg + Zn

1

Non-dialyzed Dialyzed -

P iberated

ml.

0.790 0.950 1.14 1.18 0.420 0.695 0.520 0.770 0.366 0.649 0.636 0.724 /

per cent

+25 +44 +50

+65 +24 +84

+77 +74

+101

libegtted

m7.

0.587 0.644 0.477 0.732 0.249 0.464 0.063 0.272 0.216 0.403 0.054 0.200

Change

per cent

+10

-19

t-25

+86 -75

+9

+88 -75

-7

1

Xalyeed + dialysate

P 1 liberated Change

%7.

0.700 0.903 1.04 1.06 0.279 0.549 0.448 0.804 0.320 0.545 0.470 0.611 /

per cent

+24 +43 +49

+97 +61

+187

+70 +47 +92

tivity. This is likewise true of such N compounds as choline and cholamine. Glutathione had a relatively high activity. This high activity may have been a result of the combined activity of each of the three a-amino groups in the molecule, indicating that an entirely free NH2 group is not necessary, or it may be related in some way to its -SH group. It should be noticed, however, that the high percentage activation due to zinc in the case of glutathione is due ultimately to the very marked inhibition pro-


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duced by the GSH alone and not to a higher final activity brought about by the addition of zinc to the glutathione.

TABLE II

Action of Amino Acids and Other Organic Compounds* As Zinc Coactivators for Dialyzed Intestinal Phosphatase

Substrate, fl-glycerophosphate; incubation period, 21 hours at 37”; final concentration of zinc, when added, 0.05 mu.

Casein hydrolysate

Glycine dl-Alanine Aspartate Glutamate Valine Lysine Arginine Cystine GSH @Alanine Choline Cholamine Lactate Pyruvate Glutarate Nicotinate Ascorbate

Final conomtrrttion

of organic compound

- nag. per cc.

0

0.3 1.0 7n.M 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.0 1.5 1.5 1.0 1.5 1.5 1.5 1.5 1.5 1.5

Hydrolysii of substrate

No Zn Plus Zn

per cent

13.6 13.0 11.6

per cent 10.3 27.0 33.5

10.5 26.1 13.0 25.8 12.1 20.8 13.1 19.0 12.2 20.0 11.2 21.0 12.0 18.9 12.5 24.3 6.7 21.3

13.7 15;l 13.7 14.8 13.6 15.2 14.8 12.5 13.9 13.7 13.7 13.7 13.6 12.7 12.8 12.1

-

108 190

148 100 72 46 63 88 57 94

220 9 8

10

* The organic compounds were adjusted to pH 8.5 before use.

From Fig. 4 it is evident that neither zinc nor the amino acid alanine had any effect on the pH optimum of the intestinal phosphatase.

The next point to establish was the optimum concentration of the amino acids in their action as zinc coactivators. The work with the amino acid glycine will be reported in detail. From Table III the following points are apparent. (a) The optimum


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X n+ f‘F anlne

/” x

4

._ e

Pm. 4. The pH-activity curve of dialyzed intestinal phosphatase in the presence of alanine (1 IXIM) and alanine plus zinc (0.03 mru). Substrate, &glycerophosphate; incubation period, 18 hours.

TABLE III

Optimum Concentration of Zinc and Glycine for Activation of Dialyzed Intestinal Phosphatase

Substrate, &glycerophosphate; pH, 9.2. The figures are the percentage hydrolysis of the substrate.

Time zn ConceLl- incubated tration

hrs. naM

2 0 0.0015 0.006 0.015 0.035

Glycine concentration

0.5 zmd

4.3 4.5 4.8 5.9 4.5 6.7 3.8 4.8 2.5 3.6

OItlM 0.9 InId

24 0 32.1 25.7 0.008 38.0 48.0 0.035 30.9 45.2 0.075 28.5 36.5 0.150 26.0 32.4

1.0 rnY 2.0 In* 4.0 DlM

5.0 5.9 3.9

7.9 7.7 5.7 8.6 9.5 9.3 7.5 10.0 9.2 6.7 9.9 10.4

1.8 mtd 4.5 nvd 8.7 lmd

22.0 15.0 12.5 46.5 38.1 28.0 62.0 47.1 37.5 47.0 60.8 47.3 37.2 45.0 60.0

The bold-faced figures represent the highest values found.


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zinc and optimum glycine concentrations are interrelated and depend on each other. A rough proportionality of the concentrations for optimum stimulations is 1 molecule of zinc for every 75 to 100 molecules of glycine; (b) the final optimum reached ap- pears to be about the same for all absolute levels of the zinc- glycine optimum concentrations; (c) the optimum range becomes much broader and flatter when the higher levels of glycine are used. The same general relations apply to the action of zinc and glycine on the P-glycerophosphatase of the kidney (Table IV) and bone (Table V) and the hexosediphosphatase and pyrophosphatase of the dialyzed intestinal preparations (data not shown).

TABLE IV

Effect of Zinc and of Glycine on Hydrolysis of@-Glycerophosphate by Kidney Phosphatase Preparation

The figures are the percentage hydrolysis of the substrate in 21 hours.

Zn ooncen- Non-dialyzed tration WYZyDJe

7n.M

0 0.0015 0.005 0.015 0.050

25.0 13.5 12.8 28.0 16.8 18.7 15.9 27.7 10.5 22.0 17.1 17.2 8.0 20.0 9.5 5.6 14.1 16.3

Dialyzed enzyme

Glyoine concentration

I OrnM 1 l.OrnM / 3.0rnM

As has been shown by Bodansky (13), the degree a

6.0 DIM.

10.9 13.5 16.1

17.5

activation of phosphatases by the magnesium ion is augmented by the presence of an amino acid. This is also evident from the magnesium curve of Fig. 3. A shifting of the optimum magnesium concentration with the amount of glycine in the reaction mixture is shown in Table VI.

The additions of combinations of zinc and magnesium ions or zinc and manganese ions to dialyzed phosphatases have pro- duced some interesting results. As has been shown in Fig. 3 and Table I, the ability of zinc ion to stimulate intestinal phosphatase activity disappears on dialysis, but the ability of this element to stimulate the same dialyzing phosphatase when it is


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activated by an optimum magnesium concentration does not disappear with anything like the same rapidity. With the completely dialyzed enzyme preparation the strong inhibitory

TABLE V

Action of Zinc and Glycine on Degree of Hydrolysis of fi-Glycerophosphate by Bone Phosphatase Preparations at pH 9.2

The figures are the percentage hydrolysis of the substrate.

Time Zn concen- incubated tration

lws.

2

21

n&M

0 0.0015 0.0045 0.015 0.030 0 0.003 0.005 0.025

No*- dislyeed e*ZyDle

11.3 14.6 11.2

6.5 4.4

Dialyzed enzyme


OUlt.3 1.0 nIM 3.0 nlM 6.0 nud

8.8 9.0 a.5 7.8 8.3 9.8 8.7 8.8 6.7 9.6 10.0 9.2 4.5 7.0 9.7 10.0 3.0 5.0 8.7 9.3

34.0 30.5 25.6 23.0 31.7 36.6 34.0 29.8 14.5 29.0 34.4 31.0

6.4 14.7 26.2 32.8

The bold-faced figures represent the highest values found.

TABLE VI

E$ect of Glycine on Magnesium Activation of Dialyzed Intestinal Enzyme

Substrate, fi-glycerophosphate; incubation period, 18 hours at 37”. The figures are the percentage hydrolysis of the substrate.

Mg concentration

nz‘u

0 0.04 0.10 0.40


OUIM 1.0 InM 3.0 nua

19.8 19.8 18.4 26.6 28.6 28.0 28.6 31.3 33.6 28.8 32.8 36.3

effect of zinc and the activating effect of magnesium tend to cancel each other (Table I).

In Figs. 5 and 6, the effect of combinations of zinc and manganese upon crude bone phosphatase is shown. It is apparent


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that zinc alone inhibits in the concentrations used, and that manganese alone has a slight activating effect. However, when the two ions are added together at a minimum ratio of Mn: Zn of about 10: 1, the activity of the phosphatase is brought to a point higher than by Mn alone. Thus the crude bone enzyme reacts to the two metals in a way analogous to the response observed in the incompletely dialyzed intestinal enzyme. This peculiar relation of the metal ions to phosphatase activity was used t.o establish the reversibility of the zinc inhibition. The addition

mM Zinc

FIG. 5. The effect of increasing amounts of zinc alone, and in the presence of a constant amount of Mn (0.08 mM), on the activity of crude bone phosphatase. Substrate, fl-glycerophosphate; incubation period, 21 hours.

of the stimulating ion (Mg or Mn) to the zinc-inhibited reaction mixture after a certain period of incubation resulted in the complete restoration of activity to that attained when the two ions were added from the beginning of the incubation period. These results are shown in Table VII. From this, it is evident that the responses of the incompletely dialyzed intestinal phosphatase and the crude bone enzyme are similar.

Relation of Zinc to Phosphatases in Vivo-The phosphatases of the bone and intestines of zinc-deficient animals (prepared as


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437 Hove, Elvehjem, and Hart

mM Manganese

FIG. 6. The effect of increasing amounts of Mn alone, and in the presence of a constant amount of Zn (0.04 my), on the activity of crude bone phosphatase. Substrate, P-glycerophosphate; incubation period, 21 hours.

TABLE VII

Reversibility of Zinc Inhibition of Phosphatases

Substrate, P-glycerophosphate; final concentration of ions, when added, Zn 0.05 mM, Mg 0.2 mM, Mn 0.1 mM; Enzyme A, 24 hour dialyzed intestinal phosphatase, Fnzyme B, non-dialyzed bone phosphatase. The figures are the percentage hydrolysis of the substrate.

Metals added

None 21.3 Zn 16.5

Mg Zn + Mg

29.5 29.1

10 hrs. 20 hn. 40 hrs. 70 hrs.

None 38.0 Zn 6.7

Mn 39.8 Zn+Mn 43.2

i Time incubated

0.5 hr. 1.0 hr.

33.0 22.0

Mg added 39.1 43.1

48.8 a.7

Mn added 51.1 54.0

- 2.0 hrs. 3.0 hrs.

43.1 47.5 28.5 32.0 50.0 62.1 54.1 60.2 58.2 64.5

62.8 70.0 11.3 14.0 57.0 77.5 65.0 72.5 70.0 78.0


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described by Hove, Elvehjem, and Hart (9)) and of rats on a high (toxic) zinc diet have been determined. The crude preparations were used throughout.

Zinc Dejiciency-From the data in Table VIII it can be seen that the intestinal phosphatase of the zinc-deficient rats is considerably lower than that in normal controls. There was also a slight, but possibly insignificant, lowering of the bone phospha-

TABLE VIII

Intestinal Phosphatase Content of Zinc-DeJicient and Control Rate

Substrate, glycerophosphate; enzyme, crude intestinal preparations; incubation period, 21 hours at 37”.

Group

Zinc-deficient rats 32.1 56.5 75.5 18.5 47.0 154 43.8 33.0 61.5 87.3 56.4 21.6 43.7 102 45.2 16.4 34.0 108 32.8 63.0 92.5 37.5 74.0 96.0 57.1

Average............... 27.4

Normal control rats 46.6 48.0 44.5 27.5 60.0

54.3

85.0 71.0 63.2 51.4 73.2

__ Average. 45.3 68.7

Per cent hydrolysis of substrate

T

7;::; Plus 0.2

in activity due to Zn

InM Mg

102 50.6 1.

82.5 48.1 63.0 41.5 61.2 94.0 49.5 22.0 72.0

57.6 61.4

I-

- Per cent increase

in activity due to Mg

105 51.6

107

58.0

80.4

25.0 38.0 76.6 19.8

39.8

tases. When optimum zinc is added in vitro to these crude intestinal preparation reaction mixtures, the response of the preparations from the zinc-deficient rats is much greater than that of preparations from the normal rats. This difference in the reaction to zinc in vitro cannot be due to a lower zinc content in the preparations from the deficient rats, since an entirely analogous difference exists with respect to the addition of mag-


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nesium ion, in vitro, as is shown in Table VIII. The possible explanations are that the crude preparations from the zinc- deficient rats have a lower amino acid content, or that there is a less abundant formation of intestinal phosphatase in the zinc- deficient animal. If the latter is the case, then the greater percentage activation by the metal ions may, perhaps, be related to the greater dilution of these enzyme solutions.

Rats on High Zinc Diet-Seven adult male rats were placed upon a stock ration and seven similar rats on the stock ration to which had been added 0.5 per cent of zinc (as zinc sulfate).

TABLE IX

Effect of Feeding High Zinc Diet (0.6 Per Cent Zinc As Zinc Sulfate) to Normal Adult Male Rats for .J Weeks

7 control rats 7 rata on high zinc diet

Range AWW?e Range Average

Bone ash, %. 59.6 -63.0 61.3 54.8 -59.0 56.4 Zn of dry bone, mg. per gm.. 0.20- 0.27 0.23 0.76- 1.25 0.97 Bone phosphatase, ‘% hydroly-

sis of substrate in 11 hrs.. 28.0 -34.0 31.0 46.1 -55.4 50.0 Intestinal phosphatase, y.

hydrolysis of substrate in 21 hrs....................... 13.5 -27.0 21.4 16.7 -41.2 28.7

Intestinal phosphatase after dithizone extraction. 13.5 -25.0 20.3 12.9 -30.0 21.5

Loss of activity due to dithizone extraction, %. 5.0 25.8

Bone ash, zinc content of the bones, and bone and intestinal phosphatase were determined (Table IX). In confirmation of the work of Waltner (15) a decreased bone ash was noticed when the high zinc diet was fed. Ionic zinc was removed from the intestinal phosphatase preparations by shaking with dithizone (in Ccl,) followed by pure CC14 to remove any adsorbed dithizone. The activity of t)he intestinal phosphatases after this treatment was considerably less for the rats on the high zinc diet and only slightly less for the rats on the basal diet. This indicates that the higher level of phosphatase activity in the rats on the high zinc diet was actually due to the higher water-extractable zinc content in their intestinal walls.


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DISCUSSION

The marked activation of crude intestinal phosphatase by mi- nute amounts of zinc ion has been shown to occur only in the presence of a dialyzable, organic substance which is present in the crude intestinal preparations but seemingly present to a much less degree in kidney and bone enzyme preparations. There is no evidence that this compound is in any way related to the co- phosphatase of Albers (16). It has been shown that several pure amino acids have an action equal to that of the zinc coactivator present in the crude intestinal preparation. However, it has not been established that the amino acids liberated by the autolysis of the intestinal mucosa are the only compounds with a zinc-coactivating property present in the crude preparation. In fact, the lability of a portion of the natural coactivator to strong alkali suggests the possible r81e of -SH groups. The pure amino acids do not show this strong alkali lability.

It has been consistently observed that the crude intestinal enzyme preparation gives no visible precipitate when trichloroacetic acid is added, while the crude bone and kidney enzyme preparations do. This indicates that the hydrolysis of the tissue proteins during the autolysis is more complete in the case of the intestinal preparations and strengthens the assumption that the natural coactivator is the liberated amino acids. The explanation for this greater degree of autolysis of the intestinal preparations may be related to the adherence of bits of the pancreas, or pancreatic juice itself, to the intestinal wall, thus furnishing tryptic enzymes to aid the breakdown of proteins. The effect of these tryptic enzymes could not be very great in the autolyzing or stored preparations, since the pH is too low; but during the incubation period of the phosphatase runs these tryptic enzymes can be expected, owing to the now alkaline pH, to be active and produce a certain amount of amino acids from the proteins in solution. Such an amino acid production may be a factor in the marked increase in the percentage activation by zinc and magnesium ions of phosphatase activity with time. It is also possible that the cells of the mucosa may be loaded with amino acids or protein fragments from food digestion.

Both the carboxy group and the NH2 group in the CY position are essential for zinc coactivation. Yet the wide difference in


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the concentration of the zinc and the amino acid for optimum effect makes it difficult to conceive of a final zinc-amino acid complex as being the actual activator. The fact that the zinc ion has a very broad and flat optimum when the amino acid concentration is near that of the substrate concentration suggests a possible r&e of the amino acid as a phosphate transporter or carrier in the dephosphorylation.

Lohmann and Kossel (17) have shown that zinc in a concentration of about 3 X lop3 mM stimulates the decarboxylation of pyruvic acid by yeast maceration juice plus thiamine pyrophosphate. When they used washed yeast as the source of carboxy- lase, zinc had only an inhibitory effect. Since in the preparation of the maceration juice the yeast was presumably allowed to autolyze before being ground, it undoubtedly contained a much higher free amino acid content than the washed yeast. It seems a possibility, therefore, that the same relation between zinc and amino acids holds for the activation of this reaction, as we have demonstrated here for the alkaline phosphatases.

The lowered intestinal phosphatase content (or activity) of zinc-deficient rats may be related ultimately to the previously established decrease in pancreatic tryptic activity during the zinc deficiency (9). It had also been shown previously (8) that the zinc deficiency is characterized by a lowered blood non-protein nitrogen and by an increase in the time required for an amino acid given by mouth to pass from the gastrointestinal tract to the blood stream. This delay in the absorption of amino acids may be explained on the basis of intestinal stasis resulting from the decreased pancreatic enzymes, or, if amino acid absorption should be shown to involve phosphorylation the slower amino acid absorption in zinc-deficient rats may be explained on the lowered intestinal phosphatase of these rats. Thus, the in vitro relations shown in this paper among zinc, amino acids, and organic phos- phoric esters may indicate an in viva relation between these sub- stances and the reactions governing them.

SUMMARY

1. Crude intestinal phosphatase activity is increased 40 to 100 per cent by the addition of zinc ions in vitro. Crude kidney and bone phosphatase activities are progressively inhibited by concen-


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trations of zinc of 4 X lo+ to 70 X low3 mM. This difference in response to zinc cannot be considered as an indication that intestinal phosphatase is different from the others, since after dialysis all three enzymes show a marked inhibition by zinc.

2. The readdition of the dialysate from intestinal phosphatase preparations restores the ability of zinc to activate the dialyzed enzyme.

3. The dialyzable zinc coactivator of intestinal phosphatase preparations is a product of mucosal tissue autolysis. All of the pure a-amino acids tried have the same coactivating property as the dialysate. P-Amino, keto, hydroxy, or aliphatic acids, or organic amines have little or no activity.

4. The ratio of the concentrations of a pure amino acid (glycine) to zinc ion is about 1OO:l for maximum activation. At high glycine concentrations the zinc optimum is broad and flat. At lower glycine concentrations the zinc optimum is sharp.

5. The intestinal phosphatase of zinc-deficient rats is considerably lower than that of normal controls. These results are discussed in relation to previously established disturbances in the nitrogen metabolism in the zinc-deficient rats.

BIBLIOGRAPHY

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E. Hove, C. A. Elvehjem and E. B. HartPHOSPHATASES

THE EFFECT OF ZINC ON ALKALINE

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