Inactivation of metalloenzymes by food constituents

Fd Chem. Toxic. Vol. 24, No. 9, pp. 897-902, Printed in Great Britain. All rights reserved

INACTIVATION

Copyright

OF METALLOENZYMES CONSTITUENTS

MENDEL FRIEDMAN, 0. Western

Regional

Research

0278-69 I5/86 $3.00 + 0.00 1986 Pergamon Journals Ltd

1986

Center,

0

BY FOOD

K. GROSJEAN and J. C. ZAHNLEY

Agricultural Research CA 94710, USA (Received 19 Nowmber

Service,

800 Buchanan

Street,

Berkeley.

1985)

and dehyA (CPA). The inhibition was maximal at pH 7.0 in the pH range 7-8.5. The extent of inhibition increased with time of treatment and PEAA concentration. N-AcetylPEAA did not inhibit the enzyme, suggesting that the free a-NH, group is required for inhibition. PEAA also inactivated the copper enzyme, polyphenol oxidase (tyrosinase). Comparative studies with three other inhibitors, lysinoalanine, ethylenediaminetetraacetic acid and sodium phytate, suggest that the potency of PEAA as an inhibitor of CPA is similar to that of sodium phytate. Of these four inhibitors and three thiol compounds also tested, PEAA was the least and cysteine the most effective against tyrosinase. The pattern of observations in these studies suggests differences in the mechanisms of action of the inhibitors studied. The formation of PEAA, lysinoalanine and sodium phytate in foods is of possible nutritional and toxicological significance.

Abstract-Phenylethylaminoalanine

droalanine,

(PEAA),

derived

from

biogenic

inhibited the enzymatic activity of the metalloenzyme,

phenylethylamine

carboxypeptidase

nitrophenyl phosphate, L-tyrosine and Tris base were obtained from Sigma Chemical Co., St Louis, MO. Cysteine, N-acetyl-L-cysteine, lysine, lysinoalanine, EDTA, sodium phytate and reduced glutathione were also obtained from Sigma. Phenylethylamine and N-acetyldehydroalanine were from Aldrich Chemical Co., Milwaukee, WI. Synthetic PEAA was prepared by an adaptation of the procedure described by Jones et al. (1981) as follows:

INTRODUCTION

Dehydroalanine residues in proteins contain a reactive conjugated carbon-carbon double bond and can interact with the E-NH, group of lysine to form lysinoalanine crosslinks (Friedman, Gumbmann & Masters, 1984a; Friedman, Levin & Noma, 1984b; Friedman & Noma, 1986). They can also interact with NH2 groups of biogenic amines, such as phenylto form phenylethylaminoalanine ethylamine, (PEAA) side chains (Jones, Rivett & Tucker, 1981; Tucker, Jones & Rivett, 1983). Because phenylethylamine in cheese, chocolate and certain red wines precipitates migraine attacks in dietary migraine sufferers (Chaytor, Crathorne 8c Saxby, 1975; Schwimmer, 1981), a need exists to understand the possible beneficial or deleterious consequences of removing the biogenic amine via PEAA formation in foods. Phenylethylaminoalanine and related food ingredients, such as lysinoalanine and sodium phytate, possess metal-chelating sites and could, in principle, adversely affect the nutritional quality and safety of foods by chelating essential metalloenzymes. This study was therefore designed to find out whether PEAA, lysinoalanine (LAL) and sodium phytate can inhibit zinc- and copper-containing metalloenzymes such as carboxypeptidase A (CPA) and polyphenol oxidase (tyrosinase), and to compare the relative inhibitory effectiveness of these compounds with that of the known metal chelator, ethylenediaminetetraacetic acid (EDTA).

3-(N-p- Phenylethylumino) -N -acetyl- LX-alanine (acetylPEAA). With stirring, 85.4 ml phenylethylamine was slowly added to 17.7 g Nacetyldehydroalanine dissolved in 136 ml H,O in a 500-ml Erlenmeyer flask. The resulting clear solution was placed in a 50°C water-bath for 72 hr. Excess amine was then removed by rotary evaporation, repeated washing with water and further evaporation, until the amine odour was imperceptible. The remaining yellow oil was triturated with 200ml chilled acetone to precipitate the amino acid derivative. The flask was kept in the freezer for 16 hr, the precipitate was filtered and the residue was recrystallized from a small amount of hot methanol. The yield was 13.0 g. The NMR spectrum, determined in trifluoroacetic acid with tetramethylsilane as a reference, was consistent with the assigned structure. monohy3-(N-p-Phenylethylamino)-DL-alanine drochloride (PEAA). A solution of 3 g acetylPEAA in 105 ml ~N-HC~, in a 500-ml round-bottom flask, was refluxed for 3 hr. After removal of excess HCl by rotary evaporation, the product was kept overnight in a freezer and recrystallized from 95% ethanol. The yield of PEAA was 1.2 g. The NMR spectrum in trifluoroacetic acid was consistent

EXPERIMENTAL

Reagents. Carboxypeptidase A (bovine, crystalline), alkaline phosphatase, polyphenol oxidase (from mushrooms), hippuryl-L-phenylalanine, p-

with 897

the

assigned

structure.

Elemental

analysis

898

MENDEL FRIEDMAN et

showed that the compound crystallized as a monohydrochloride (found: C, 53.9; H, 7.06; N, 11.4; Cl, 14.2+alc. for C, ,H,,N,O,CI (mol wt 244.5): C, 54.0; H, 6.94; N, 11.4; Cl, 14.5). Carboxypeptidase assay. CPA activity was assayed by the method of Folk & Schirmer (1963) using hippuryl-L-phenylalanine as substrate. The substrate (3.26 mg) was dissolved in 100 ~1 95% ethanol and diluted to 10 ml in pH 7.5 Tris HCl (25 mM containing 0.5 M-NaCl), and 2.9 ml of this substrate solution was pipetted into a cuvette and incubated at 25°C for 3 min in a Cary 14 spectrophotometer with temperature control. To this substrate solution was added 0.1 ml of the enzyme solution, prepared by dissolving IO ~1 CPA in 2 ml 10% LiCl. The commercial CPA stock solution contained approximately 1 IU/pl. The resulting increase in absorbance at 254 nm was measured for 1 min. Enzyme activity was calculated as described by Worthington Diagnostic Systems (1982).

C,HS-[CH,],-NH,

al.

in 1 ml H,O and the mixture was incubated for 15 or 60min at 25 or 37°C. At the end of the reaction, 100~1 of the solution was pipetted into 2.9 ml of oxygen-saturated substrate solution and the remaining activity of the tyrosinase was determined as described above. Details of the inhibitors and reaction conditions for specific experiments are given with the results. RESULTS

Formation

AND DISCUSSION

of PEAA

Equations (1) and (2) illustrate the formation of PEAA from phenylethylamine and Nacetyldehydroalanine. The amine participates with the double bond of dehydroalanine in a nucleophilic addition to form acetylPEAA (equation 1). Hydrolysis with HCI produces PEAA (equation 2). Since a new asymmetric carbon is created during the addition, synthetic PEAA consists of an equimolar mixture of D- and L-enantiomers.

+ CH, = C(NHCOCH,)-COOH --+ C,H,-[CH,],-NH-CH,-CH(NHCOCH,

)--COOH

(1)

AcetylPEAA AcetylPEAA

+ HCl -+ C,H,-[CH*],-NH-CH,-CH-COOH

(2) I

PEAA Effect of PEAA or acetylPEAA on CPA. In a typical experiment, 5.2 mg (21 pmol) PEAA was dissolved in 1 ml 25 mM-Tris buffer, pH 7.0, 0.5 M-NaCl. To this solution was added 5 ~1 of undiluted CPA solution. The solution was then incubated at 37°C for 1 hr. For acetylPEAA, the conditions were the same, except that 84 pmol acetylPEAA replaced the PEAA. Alkaline phosphatase assay. Either 21 or 42 pmol PEAA was dissolved in 1 ml 0.75 @-alanine with 50 mM-NaCl, pH 10.2. After addition of 50 ~1 of the enzyme stock solution (1 mg/ml), the mixture was incubated at 37°C for 1 hr. Alkaline phosphatase activity was determined as described by Worthington Diagnostic Systems (1982). Polyphenol oxidase assay. This assay was carried out as described by Worthington Diagnostic Systems (1982) with some modifications, as follows: 1 mg tyrosinase containing 2000 U was dissolved in 1 ml of deionized distilled water and diluted to a concentration of 400 U/ml H,O. A mixture of 1 ml 0.5 M-phosphate buffer, pH 6.5, 1 ml 1 mM-L-tyrosine and 0.9 ml H,O was oxygenated by bubbling oxygen into the solution through a capillary tube for 5 min. This oxygen-saturated substrate solution was transferred to a cuvette, which was placed in the spectrophotometer, and the A,,, values were recorded for 5 min at 20’C until equilibrium was reached. To the substrate solution was then added 100~1 of the enzyme solution and the AZROvalues were recorded for 12 min. Enzyme activity was calculated by: Activity Eflect inhibitor

(Ujmg) =

A,,,/min

x 1000

mg enzyme in reaction mixture

of inhibitors.

An appropriate amount of the was mixed with 200 or 400 U of the enzyme

NH,. HCl

Inactivation

of carboxypeptidase

A

The influence of several parameters expected to govern the inhibition of CPA by PEAA was explored to define the scope of the reaction. Table 1 summarizes the extent of inhibition of CPA by PEAA in Tris buffers of pH 7.0, 7.5, 8.0 and 8.5 at 25 and 37°C. Since inhibition within the range studied was highest at pH 7, this pH at 37°C was adopted as standard for most of the other studies. A series of experiments using reaction times of 3&480 min was carried out to assess the influence of time on the extent of inhibition of CPA by PEAA. Under the conditions used (21 pmol PEAA and 5 ~1 (5 IU) CPA/ml at pH 7.0 and 37°C) inhibition increased with time, being 54.3, 64.9, 77.1, 89.8 and 97.2% after exposure for 30,60,120,240 and 480 min respectively. For convenience, a 60 min exposure was adopted as standard for most of the experiments.

Table I. Inactivation of carboxypeptidase A (CPA) with phenylethylaminoalanine (PEAA) Temp. (’ C) 25

31

PH 7.0 7.5 8.0 8.5 7.0 7.5 8.0 8.5

CPA inhibition (%) 83.7 43.5 38.0 28.3 100.0 100.0 79.6 61.2

Incubation conditions: X4 pm01 PEAA and 5 ~1 (5 IU) CPA/ml for I hr. No inactivation was observed in enzyme controls without PEAA.

Metalloenzyme

inactivation

Five experiments were carried out to assess the influence of PEAA concentration on the extent of inhibition of CPA, with concentrations of PEAA ranging from 0.84 to 84mM. In incubations of 1 ml PEAA solution with 5 ~1 undiluted CPA at pH 7.0 and 37°C for I hr, the extent of inhibition markedly increased with increasing PEAA concentration, being 3.8, 29.6 and 66.8% with concentrations of 0.84, 8.4 and 21 .O mM respectively and reaching a maximum (100%) at about 42 mM PEAA. In contrast to the 100% inhibition effected by 84 mM-PEAA, the degree of inhibition by the same concentration of acetylPEAA was only 1.05%. Since CPA is one of several zinc enzymes, alkaline phosphatase being another, it was of interest to find out whether PEAA would inhibit other members of this class. We found that under the conditions described in the Experimental section, 82.2% of the phosphatase was inactivated or inhibited with 21 pmol PEAA and 88.8% with 42pmol. These results suggest that PEAA may be a genera1 inhibitor of zinc enzymes. We have previously reported that sodium phytate has a small inhibitory effect on CPA (Friedman, Grosjean & Zahnley, 1985). Additional experiments were carried out to delineate further the inhibitory action of sodium phytate on this enzyme. CPA was exposed to a series of concentrations of sodium phytate ranging from 1 to 50 mM. Enzyme activity was measured at three substrate concentrations: 0.2, 0.5 and 1 mM. Table 2 shows that the inhibitory activity of the phytate salt depends on the concentration of both the inhibitor and the substrate. The data also show that the threshold concentration for observable inhibition is between 1 and 10 mM-sodium phytate. The potency of sodium phytate as a CPA inhibitor is similar to that of PEAA and much lower than that of LAL. Studies on the relative effects of three inhibitors (PEAA, LAL and EDTA) on CPA activity at three substrate levels are plotted in Fig. 1. Because of PEAA’s low inhibitory potency at 25”C, the PEAA experiments were carried out at 37°C. In contrast, because of rapid inactivation of CPA by LAL at 37’C, these experiments were carried out at 25°C. The data for the known metalloenzyme inhibitor, EDTA, obtained at 25°C are included for comparison. Additional studies showed that inhibition by EDTA at 37°C was greater than that shown at 25”C, reaching 84% at 0.5 mM-EDTA and 100% at 1.5 mM. These observations show that CPA inactivation is influenced by the concentration of the inhibitors and

Table

2.

Etkct

peptidase

of

A (CPA) CPA

Na

sodium

phytate

activity

inhibition

concentration

at three (%)

on

carboxy-

substrate

concentrations

substrate

concns

with

(mu)’

of:

phytate

concn

(ItIM)*

0.2

0.5

0

0

1.0

f

3.16

30.1

f

1.25

42.6

i

I5

38.3

+ 2.37

44.3

f

1.25

55.4

i_ 0.69

25

55.0

k 2.36

56.6

+ I .30

70.6

k 0

66.2

i

59.1

*

X0.3

k 0.80

Values

are

corrected

conditions: means for

0.0 37 C for

+SD any

for loss

two

3.04

2.08

60 min. separate

of activity

in the

3 60

.o = 6 G ‘-

40

$ 0

Concn

of

lnhlblror

ImM)

(b) 70

OLI 0

I 5

I 10 Concn

of

I 20

I 15 InhIbItor

I 25

(mM)

Fig. 1. Inhibition of carboxypeptidase A (CPA) activity by (a) c2.5 mh+ethylenediaminetetraacetic acid (EDTA); 0, a, 0) and I-Smwlysinoalanine (LAL; W, 0) and (b) g2.5 mM-EDTA (m, A, 0) and 1&25mwphenylethylaminoalanine (PEAA; 0, A, O), expressed in both graphs as a function of the inhibitor concentration and the substrate concentration (0.2 (0, n ), 0.5 (A, A) and 1.0 (0, 0) mM substrate). Incubation conditions: for EDTA60min at 25°C; for LAL-5 min at 25°C; for PEAA60min at 37°C.

raise the question as to whether the reported adverse effects of protein-bound phytate on mineral nutrition (Clydesdale & Camire, 1983; Graf & Eaton, 1984) could be partly due to the inactivating effect of chelation of bound phytate to metalloenzymes. Inactivation of polyphenol oxidase (tyrosinase)

0

31.7

50

899

1.0

IO

*Incubation

by food constituents

determinations enzyme

controls.

and

are

Experiments were also carried out to ascertain whether LAL, PEAA, EDTA and sodium phytate can inactivate the copper-containing enzyme polyphenol oxidase (tyrosinase). The enzyme was exposed to three concentrations (0.4, 1.O and 2.5 mM) of each of these compounds at 37°C for two periods (15 and

MENDEL FRIEDMAN et al.

900 Table 3. Effects of time and concentration

on the inactivation of polyphenol oxidase (tyrosinase) by four inhibitors at 37’C Tyrosinase inhibition (%) with inhibitor concns (mM) of: 1.0

0.4 Inhibitor

Incubation time (min)

LAL Na phytate EDTA PEAA

2.5

15

60

15

60

15

60

loo+0 62. I i 4.88 39.4 f 0 25.0 + 2.36

100 io 84.5 k 2.44 74.2 k 2.14 28.3 + 7.07

100+0 75.3 + I.83 51.9i 1.84 37.6 i 0

100 *o 98.7 k 1.84 74.0 * 0 42.9 k 7.35

loo*0 93.8 f 0 70. I + 0 57.8 k 2.21

loo+0 loo*0 72.5 + 3.54 loo*0

LAL = Lysinoalanine EDTA = Ethylenediaminetetraacetic acid PEAA = Phenylethylaminoalanine Values are means *SD for two separate experiments. Enzyme controls run simultaneously showed no loss in activity after I5 or 60 min.

Table 4. Effect of temperature and of concentration of inhibitor and substrate on the inhibition of tyrosinase by sodium phytate Tyrosinase inhibition (%)t by sodium phytate concns (mu) of: Substrate concn (InM) 1.0

0.25 1.0

Incubation temD. (’ cl* 25 37 47 25 25

01 0.0 35.9 53.1 0.0 0.0

0.25

0.50

Experiment A 33.8 81.3 100.0

1.0

72. I

100.0 100.0

Experiment Bf 0.0 26.6 + 9.33

65.0 k 7. I 66.0 + 8.51

90.0 * 0 78.0 k 2.8

*Incubation with 200 U tyrosinase for I hr. t(Activity of enzyme plus sodium phytate/activity of enzyme alone) x 100. ZResults are means *SD for two separate experiments.

60 min). Table 3 shows that LAL is a potent inhibitor of tyrosinase, completely inactivating the enzyme under all conditions tested. Sodium phytate appears to be next in potency, a concentration of 0.4m~ inactivating 62% of the enzyme in a 15-min exposure and 84.5% after 60min. The corresponding values for 1 mM-phytate are 75.3 and 98.7% and for 2.5 mM, 93.8 and lOO%, respectively. Thus inactivation is both concentration and time dependent. The next most effective inhibitor was EDTA followed by PEAA, the least potent of the four tested. In both of these cases, the extent of inactivation was also a direct function of inhibitor concentration and time of exposure. Additional experiments, summarized in Table 4, show that the extent of inactivation of tyrosinase is strongly dependent on the concentration of sodium phytate and to some extent on the concentration of the substrate. Generally, in classical enzyme-inhibitor interactions (Segel, 1975), the higher the substrate concentration, the lower the inhibition. Since our data show that this is apparently not the case for the inhibition of tyrosinase by sodium phytate, the apparent inactivation appears to be the result of a combined effect of inhibitor and substrate. The data also show that sodium phytate inhibits the copper enzyme (tyrosinase) more effectively than the zinc-containing (CPA). The results listed in the Tables and in Fig. 1 suggest that LAL, EDTA and PEAA are also more effective as inhibitors of tyrosinase than of CPA. Table 5 shows that thiols such as cysteine and N-acetyl-L-cysteine are potent inhibitors of tyrosinase. In contrast, lysine up to a IO-mM concentration had no measurable effect on the activity of tyrosinase.

A series of experiments was also carried out to find out whether CuSO, would alter or reverse the inhibition of tyrosinase by LAL, PEAA, sodium phytate and EDTA. The results (Table 6) varied with the inhibitor. For LAL, inhibition was not affected by adding the copper salt after the enzyme was inactivated (sequential addition), by exposing the enzyme to LAL and copper sulphate added simultaneously, or by exposing the enzyme to the inhibitor plus copper sulphate (reverse addition). For PEAA and EDTA, the presence of copper sulphate enhanced the inhibition under all three conditions, while for sodium phytate, the presence of the copper salt lowered the extent of inhibition with both the simultaneous and reverse additions. The latter results imply that copper ions bind or chelate to the inhibitory sites of sodium phytate, lowering its inhibitory potency. Evidently, the copper of the active site of the enzyme and added copper compete for metal-chelating sites on sodium phytate. Mechanistic Figure

possibilities 1 suggests

that

the patterns

of enzyme

Table 5. Inhibition of tyrosinase by L-cysteine, N-acetyl-r-cyst&e and reduced glutathione Tyrosinase inhibition (%)’ with inhibitor concns (mM) of: Inhibitor

0.05

0.1

0.5

L-Cyst&e N-Acetyl-L-cyst&e Reduced glutathione

0.0 0.0 0.0

37.5; 39.1 25.0; 25.7 12.5; 13.0

100.0; 100.0 100.0; 100.0 100.0; 100.0

*Incubation conditions: 200U tyrosinase with the indicated inhibitor concentrations at 25°C for 60 mm. Values are results from two separate experiments.

Metalloenzyme

Table

6. Effect

of copper sulphate (PEAA),

on the inhibition

sodium

by food constituents

inactivation

phytate

of tyrosinase

by lysinoalanine

and ethylenediaminetetraacetic

(LAL),

LAL

Tyrosinase

+ inhibitor.

I hr

Tyrosinase

+ inhibitor,

I hr; then CuSO,,

(sequential Tyrosinase

+ CuSO,,

(reverse Tyrosinase

addition) I hr:

then tyrosinase,

100 *o

lOOi_

100 *o

loo*0

loo*0

I hr

200 U tyrosinase,

In control

91.2iO

100 +o

loo+0

experiments

loo*0

4.3 +6.l 0.5 rn~

it was found

57.6 k 0.5

100~0

I hr

addition) + inhibitor?,

*Conditions:

EDTA

100+0

50 * 0

+ CuSO,,

I hr (simultaneous Inhibitor

(%)

Na Dhvtate

5 min

addition)

+ inhibitor

inhibition

PEAA

100 *o

phenylethylaminoalanine

acid (EDTA)

Tyrosinase Conditions*

901

inhibitor

except where

54.3 i indicated

that in the absence of inhibitors,

3.

I

42.5 + 0.5

lOOi_

3X.8 k 5.7

17.4k6.1

otherwise

(7) and 0.5 mwCuS0,

tyrosinase

lost

16.2%

of its actiwty

at 37 C. in I hr

at 37’C. tlnhibitor Values

concn reduced from are means

*SD

for

0.5 to 0.1 mht.

two determinations.

inhibition differ with these three inhibitors. Moreover, inhibition of CPA by LAL reaches equilibrium in about 3 min but complete inhibition by PEAA requires about 1 hr. Concentration effects do not show simple behaviour. LAL displays sharp ‘titration’ of CPA activity between about 2 mM (threshold) and 4m~. EDTA is inhibitory at even lower concentrations than LAL. PEAA inhibition of CPA was more concentration-dependent above 15 XtM, suggesting two classes of inhibiting sites. Effects of substrate concentration (over a fivefold range) for both LAL and PEAA are complex and not clear-cut. Differences in the shapes of the inhibition curves suggest that no single mechanism is operating (Segel, 1975). Differences in the mode of action of the three inhibitors seem reasonable, because structural features differ. Thus, EDTA may act primarily by chelating to the zinc or copper of the enzymes, while the action of LAL may be complex and depend on pH (Friedman et al. 1985) and PEAA, unlike the others, has a Ph---CH,-CH,-moiety that makes it resemble CPA substrates in structure. Our conclusions about the mode of inhibition of carboxypeptidase are consistent with those proposed by Charlier, Dideberg, Jamoulee et al. (1984) for the zincinactivation of the active-site-directed carboxyD-alanyl-D-alanine-cleaving containing peptidase of Streptomyces albus G. These authors report that, depending on the structure of the inhibitor, enzyme inhibition may be competitive (with binding occurring at the active site) or noncompetitive (with binding causing disruption of the protein crystal lattice). Additional studies showed that acetylPEAA does CPA (1.05% inhibition by inhibit not 84m~-acetylPEAA compared with 100% by 84 mM-PEAA). This fact suggests that the free a-NH, group in the alanine part of PEAA is required for inactivation of CPA, and that the amino group probably interacts with the active site of the enzyme during the inhibition process. Possible nutritional

and toxicological

sign$cance

The formation of PEAA in foods may also have therapeutic and toxicological connutritional, sequences. First, formation of PEAA from its biogenie amine precursor may decrease the migrainecausing potential of phenylethylamine (Chaytor et al.

1975; Ingles, Back, Allimore et al. 1985; Ingles, Tindale & Gallimore, 1978 & 1980; Luthy & Schlatter, 1983). This could be beneficial, provided PEAA itself does not turn out to be deleterious. Secondly, inhibition of CPA by PEAA, LAL and/or sodium phytate in uivo could affect the digestion of foods, since CPA participates in the removal of C-terminal amino acid residues during the digestion of proteins (Schwimmer, 1981). These considerations also imply that in viuo chelation of the inhibitors to metalloenzymes and essential trace elements could affect mineral nutrition, provided the inhibitors are present at high enough concentrations in foods. This is especially true for sodium phytate, which is known to affect the nutritional quality of foods adversely (Clydesdale & Camire, 1983; de Rham & Jost, 1979; Graf & Eaton, 1984; Knuckles, Kuzmicky & Betschart, 1985; Rodriguez, Morr & Kunkel, 1985). The described inhibition of zinc- and coppercontaining metalloenzymes by sodium phytate implies that dietary phytate can affect mineral nutrition by chelating to essential trace elements such as zinc, copper and iron (Henkin, 1974); Lonnerdal, Keen & Hurley, 1984). It can also alter the activity of important classes of enzymes needed for normal metabolic activity. The possible consequences of such inhibition for nutrition, food safety and health await further study. Since polyphenol oxidase catalyses oxidative browning in many plant foods (Deshpande, Sathe & Salunkhe, 1984; Friedman & Smith, 1984a,b; Hurrell & Finot, 1984), efforts have been made to inactivate the enzyme in order to minimize possible undesirable deteriorative and antinutritional effects associated with such browning reactions (Golan-Goldhirsh & Whitaker, 1984). The results of the present study suggest that the tyrosinase inhibitors listed in Tables 3-5 should also be investigated for their ability to prevent oxidative browning in foods. Finally, our findings also suggest a possible explanation for the reported LAL-induced cytomegaly in the proximal tubules of the rat kidneys (Finot, 1983; Gould & MacGregor, 1977; Pfaff, 1984; Woodard, Short, Alvarez & Reyniers, 1975), since they support the hypothesis (Friedman, 1977; Friedman et al. 1984a; Hayashi, 1982) that binding of LAL to metalcontaining proteins, such as metallothioneins in the kidney cells, could provide a molecular basis for this observed biological effect.

MENDEL FRIEDMAN et al.

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