Intestinal dipeptidases I. Spectrophotometric determination and characterization of dipeptidase activity in pig intestinal mucosa

149

BlOCHIMICA ET BlOPHYSICA ACTA BBA

65235

INTESTINAL DIPEPTIDASES 1. SPECTROPHOTOMETRIC DETERMINATION AND CHARACTERIZATION

OF DIPEPTIDASE ACTIVITY IN PIG INTESTINAL MUCOSA

LARS JOSEFSSON AND TOR LINDBERG Departmen; of Physiological Chemistry, University of Lund, Lund, Sweden (Received January 26th, I965)

SUMMARY 1. A new simple and rapid spectrophotometric assay for dipeptidase (dipeptide hydrolase, EC class 3+3) activity is described. 'The method is based upon the decrease of absorption at 220 mp, as peptide bonds are hydrolyzed. By using ethanol as a precipitating reagent, the method is suitable for the study of peptidase activity in crude tissue extracts or partially purified preparations. The accuracy is about ± I %' and the sensitivity is greatly increased as compared with procedures commonly used hitherto. 2. The method has been employed for the determination and characterization of r.-alanyl-t-glutamic acid, glycylglycine, glycyl-t-leucine and glycyl-L-valine hydrolysing activities of pig intestinal mucosa. 3. The results suggest that the four different activities are related to four separate enzymes. 4. The relation of our findings to previous knowledge of the properties of the dipeptidases has been discussed.

INTRODUCTION

The localization and function of enzymes in gastric and intestinal mucosa has attracted greatly increased interest during the last few years->. At least part of this interest has been stimulated by recent studies of different malabsorption syndromes with results suggesting the lack of necessary enzyme systems-.s. In spite of the increasing amount of information obtained during recent years about the digestion and absorption of different nutrients under normal and pathological conditions, little has been obtained about the final digestion and absorption of proteins, i.e. peptides and amino acids. Such studies have been greatly hampered by the lack of suitable methods for assaying the dipeptidases (dipeptide hydrolases, EC class 3.4.3)5. The methods in use" require relatively large amounts of substrate, are laborious and are inadequate for the study of dipeptidase activities in crude tissue extracts or partially Biochim, Biophvs. A eta, I05 (I965) r 49-J6I

ISO

L. J OSEFSSON, T. LI NDBERG

purified preparations. We therefore attempt ed t o find a simple and rapid proc edure whi ch would be suitable for such studies. Since it was r ecent ly report ed that dipeptides, in contrast t o most ofthe amino acids, exhibit a strong abs orpt ion in the low ultraviolet region ", it was obvious to att empt to utilize t his difference in absorption t o follow the dip eptidase activity. H owever, interference by substances, such as proteins, carb oxylic acids and buffer ions which also absorb strongly in th e low ultra violet region and which are pr esent in relati vely high concentration in crude tissue extracts was found to nullify any advant age over othe r methods which are based on the release of amino groups ?" or ca rboxyl groups !' . Simila r findin gs have been report ed by others s-", It was therefore n ecessary to employ a reagent which removes these substan ces wit hout influencing the dipeptide or amino acid residues. Ethanol, with a negligible absorption down to 210 mil, was found t o be suitable for this purpose and allowed the strong absorption of the peptide bond t o be fully utilized P. The present paper r eports the details of the proc edure and its employment in t he determination and characterization of several dipeptidase activities in the inte st inal mucosa of t he pig. EX I'ERIMENTAL

M ethods T ot al nitrogen was determined by the micro-Kj eldahl procedure with mercuric oxide as catalyst. Paper chromatographic an alysis were m ade on Whatman No. I filter paper usi ng n-butanol-acetic acid-wate r (4 : I : I vfv) as solvent. The spot s were visualized by sprayi ng wit h ninhydri n. Con striction mi cro-pipettes were used throughout for volumes less then I ml. For larger volumes, a cons tant delivery aut omatic p ipett e was used. Th e mixing of sa m ples was performed with th e aid of a vib rator (Cycle-Mixer]. Absorbancy was measured with a Beckman DU spectrophoto me ter p rovided with photomultiplicator and 1 -cm semi-m icro cuve ttes. Mucosal extracts we re prepared at 4° by scraping the muco sa from r-c-cm lengths of the pig intestine. The scrapings were cooled in an icebath and homog enized for 2 min in 2 ml of 0.1 M sodium chloride solution at 14500 rev.fmin using an MSE homogenizer. The homogenat e was left at 4° for 3 0 min to p ermit quantitative extraction and was then centrifuged for 30 min at 27 000 X gat 4° in an International high speed centrifuge, Model HR 1. The clear sup ernatant was diluted with water according to its t otal nit rogen content and used dir ectl y as the enzyme solution . M aterials Intestine from adult pigs was used throughout. The pigs had been starved for 24 h b efore death to ensure an empty intestine . The intesti ne was immediately removed , chilled in ice and t he mucosal extracts were prepared within I h after death. L-A lany l-L-glutamic acid·H20 (Yeda, Rehovoth, Israel , Lot No. ALGL I). An alysis; found: N, 12.1 ; CsH14 NPs·H 20 (236.2) requires N, II .8 ; [a]24 _8° in wat er (c, 1 .0 ); found t o be chromatographically pur e, Rp 0. 23. A 0 .03 M aqueous solut ion served as dipeptide solution. B i ocbim . B i ophy s. Acta, 105 (I g 65) 14 9-1 61

INTE STINAL DIPEPTIDASE . 1.

Glycylglycine (NBC, Cleveland, Ohio, Lot No. 1973). Analysis; found : N, C4H sN20 3 requires N, ZI.Z ; found to be chromat ographically pure , R p O.l Z. A 0.075 M aqueous solution serve d as dipeptide solution. Glycyl-i-leucine (NBC, Cleveland, Ohio, Lot No. 4451). Analysis ; found: N, 15. 0 ; CSH16N20 3 (188 .2) requires N, 1+ 9 ; found to be chroma togr aphically pure, Rp 0-46. A 0.0375 M aque ou s solut ion ser ved as dip ept ide solut ion. Gly cyl-L-valin e (Yeda, Rehovoth, Isr ael. Lot No. GOLK). Analysis ; found: N, 16.1; C7H u N 20 3 (174.2) requires N, 16.1 ; [aJ24 -20 in water (c, 1.0); found to be chromatogr aphically pure, R p 0.37. A 0.03 M aqueous solution served as dip eptide solution. A mino acids. Th ese were commercial produ ct s of Man n Research Lab s., New York. All specimens gave satisfactory analysis for nitrogen and were found to be chromatographicall y pur e. They were used in aq ueous solutions in combinations and concentrations as follows : 0.03 M L-alanine and 0.03 M t-glutamic acid; o.IS M glycine; 0.0375 M glycine and 0 .0375 M L-leucine; 0.03 M glycine and 0.03 M L-valine. Buffer solutions. All Duffers were made 0.15 M. Th e borate stock buffer was prepar ed according to CLARK and the phosphate st ock buffer according to S0REN SEN. Appropriate pH values (glass elect rode) were obtained by adjusting the stock buffers wit h 1 N NaOH solu tion and diluting with water. Th e pH values for t he digest mixtures were checked before and after digestion. S alt soluti ons. Aqueous stock solutions of CoC12 , MgClz, Mn(C2H g0 2)2 and ZnC1 2 (an alytical gra de) wer e m ade at a concentration of 0.002 M. Appropri ate dilutions were made wit h the buffer solutions t o give a final buffer concent ration of 0. 15 M and a concentration of the salt ranging from 10-4 t o 10- 6 M. Ethan ol-water (99:1 v /v), having a negligible absorption down to Z10 mtt , was used as pr ecipitating reagent . Glass-distilled wat er was used throughout. ZI. O ;

0

RESU LTS

Standard procedure The assay was carri ed out in conical centrifuge tubes with a capacity of about IS ml. 50 ,ul of the dip eptide solution and roo ttl of the buffer solution were mixed in the assay tubes, which were th en placed in a wat erb ath at 40 ± 0.050 and equilibrated. At zero time 20-tt l samples of enzyme soluti on were pipetted directly into the subst rat e solution and thoroughly mixed. After suitable incubat ion time the hydrolysis was interrupted by t he addit ion of 1.3 ml of pre cipit ating reagent and mix ed thoroughly with the incu bation solution with th e aid of t he vibrat or. After st anding for ro min the precipitat e was cent rifuged down in an angle centrifuge, Wifu g Model X I , at 5 0 0 0 rev .Jmin for 20 min . A I -ml sample of the sup ernatant was transferred to th e semi- micro cuvette and the absorba ncy measured at 220 mtt . Id en tically t reat ed samples contai ning So III of the corresponding amino acid soluti on inst ead of the dip eptide solution serve d as blanks. The absorb an cies of the different incubation mixtures when made according t o the standard assay proc edure and precipit ate d at zero time are given in Figs. 1-4. An extract of mu cosa t aken 100 em from the pylorus served as enzyme solution. As seen from the difference spectra in the figures, the peptide bon d absorp tion obtained for Biochim. Biophy s. A cta, raj (1965) I4 9-161

L. JOSEFSSON, T. LINDBERG

2.0

2

2.0

e

>-

c: e .a

u

s .c

6 1.0

61.0

.8

«'"

«

.a

250

225

250

225

mf

mf

Fig.!. Absorbancy of digest mixtures containing r.5/tn~oles of i-alanyl-t-glutamic acid (---) and I.5/lmoles of t-alanine and LSI/moles of L-glutamic acid (.-.) at zero time by the standard assay procedure. Mucosal extract added contained 4.23/lg of nitrogen. Borate buffer (pH 7.4). - - - , difference spectrum. Fig. 2. Absorbancy of digest mixtures containing 3.75/lmoles of glycylglycine ( - - - ) and 7.5 /Imoles of glycine (.-.) at zero time by the standard assay procedure. Mucosal extract added contained r6.3/1g of nitrogen. Borate buffer (pH 7.9). - - - , difference spectrum. 4

2.0

>-

u

c: e .o L.

~

1.0

«

225

250

225 n~

mf

Fig. 3. Absorbancy of digest mixtures containing 1.88 pmoles of glycyl-t-Ieucine (---) and r.88llmoles of glycine and 1.88 flmoles of r.-leucine (. -.) at zero time by the standard assay procedure. Mucosal extract added contained 2·58 flg of nitrogen. Borate buffer (pH 7.9). - - - , difference spectrum. Fig. 4. Absorbancy of digest mixtures containing L5/lmoles of glycyl-L-valine (---) and 1.5 flmoles of glycine and 1.5 pmoles of r.-valine (. -.) at zero time by the standard assay procedure. Mucosal extract added contained 3.25 Ilg of nitrogen. Borate buffer (pH 7.6). - - - , difference spectrum.

Biochim, Biophys. Acta, lOS (1965) 149-16r

153

INTEsnNAL IlIPEPTtDASES. 1.

the dipeptides in the concentration used in the assay accounts for an absorbancy of about 0.8 at 220 mfl. The figures also demonstrate the efficiency of using ethanol as a precipitating reagent. The low absorbancy of the samples containing the amino acid solutions permits a narrow slit width of the spectrophotometer when they serve as blanks and eliminates the risk of interference from stray radiation occurring in the low ultraviolet region 13. Determination oj the time course oj hydrolysis The rates of hydrolysis of the four different dipeptides were studied by following the decrease in the peptide bond absorption after suitable time intervals. The 6

5

1.0

1.0

""E

}

o

...~

o

0.5

~

0.5

""

o L_-,-_-:,:--2:::::::::r:::2==!L.J o

10

20

0 '---'---'---'_"'---'---'-_'---'--'------'. 0 50 100

min

min

Fig. 5. Time-curve for the hydrolysis of z.-alanyl-t-glutamic acid by pig intestinal t-alanyl-rglutamic acid dipeptidase added in mucosal extract containing 15.8 fig of nitrogen. Borate buffer (pH 7.4). Fig. 6. Time-curve for the hydrolysis of glycylglycine by pig intestinal glycylglycine dipeptidase added in mucosal extract containing q·4l1g of nitrogen. Borate buffer (pH 7.9). 8

7

~

E 0.5

o

gj

""

O'--_ _--L o 5

-'-

10 min

'--...:::;:,~-o----'

15

20

0'--_ _---'

o

-'20

10

-'---' 30

min

Fig. 7. Time-curve for the hydrolysis of glycyl-t-leucine by pig intestinal glycyl-L-leucine dipeptidase added in mucosal extract containing 1.44 fig of nitrogen. Borate buffer (pH 7·9). Fig. 8. Time-curve for the hydrolysis of glycyl-t-valine by pig intestinal glycyl-L-valine clipeptidase added in mucosal extract containing 2.87 fig of nitrogen. Borate buffer (pH 7. 6).

Biochim, Biophys. Acta, 105 (1965) 149-161

154

L. JOSEFSSON, T. LINDBERG

results are shown in Figs. 5-8, where the activity, LlA 220 , is plotted as a function of time. The hydrolysis of each of the four dipeptides closely followed a linear function of time at the beginning of the reaction. At later times a striking difference was apparent. Thus, when 30-40% of the glycylglycine and glycyl-L-leucine had been hydrolyzed the rate began to fall off. In contrast to this, the hydrolysis of L-alanylL-glutamic acid and glycyl-L-valine continued to be linear with time up to 60-70% completion. The assay procedure permitted each of the reactions to be studied to completion.

--;l

10

I

0.02

0.1 c

-t.}

t:

\:

o

.....,:::Jam

~ 0.05

.....,'"

Nitrogen

00 I<:~----=------!=----' 10 5 Nitrogen Cf g)

'f g )

Fig. 9. Pig intestinal L-alanyl-L-glutamic acid dipeptidase activity venus enzyme concentration expressed as the amount of total nitrogen added. Phosphate buffer (pH 7.4). Fig. to. Pig intestinal glycylglycine dipeptidase activity versus enzyme concentration expressed as the amount of total nitrogen added. Borate buffer (pH 7.9).

11

0.1

12

0.1

c

'E ~ E

0

....

gj 0.05

0l<-_-'-_-,"_ _-'-_-'-_ _L.......1

o

5 Nitrogen 'fg)

0 0

5 Nitrogen(f g)

Fig. I I. Pig intestinal glycyl-r-Ieucine dipeptidase activity versus enzyme concentration expressed as the amount of total nitrogen added. Borate buffer (pH 7.9). Fig. 12. Pig intestinal glycyl-L-valine dipeptidase activity versus enzyme concentration expressed as the amount of total nitrogen added. Borate buffer (pH 7.6). Biochim, Biophys. Acta, 105 (1965) 149-161

INTESTINAL DIPEPTlDASES. 1.

155

I nfiuence oj enzyme concentration The relationship between activity and enzyme concentration was studied by adding the enzyme solution made up in a serial of different dilutions and measuring the decrease in absorbancy after a certain time interval. Each of the four dipeptidase activities displayed a linear correlation with enzyme concentration (Figs. g-12). With the enzyme concentration expressed as amount of total nitrogen, however, it was found that the rate of the different reactions differed greatly. Thus, when I ,amole of glycylglycine was hydrolyzed about 4 p.moles of t-alanyl-t-glutamic acid, 8,amoles of glycyl-t-leucine and 7 [lmoles of glycyl-tvaline were hydrolyzed during the same time. Determination oj the pH optimmn The effect of pH was studied by measuring the activity of the four dipeptidases over a pH range from 6 to g. The pH of the reaction mixture was checked with the glass electrode. The four dipeptidases were found to be pH-sensitive over the range studied showing rather sharp maxima (Fig. 13). Their pH optima differed slightly from

VI

40

';)i

2:0

<.

'0

'"

:I:

f

20

o '-0---'-0,<:;....----'----...1---_ 6

8

7

9

pH

Fig. 13. pH-activity curves of pig intestinal dipeptidases measured by the standard assay procedure. e-e, mucosal extract containing 0.44 pg of nitrogen added to L51tmoles of r.-a.lanyli.-glutamic acid, 10 min digestion; 0-0. mucosal extract containing 7.8 pg of nitrogen added to 3.75 pmoles of glycylglycine, IS min digestion; . - . , mucosal extract containing 0.83 fig Itg of nitrogen added to 1.88,umoles of glycyl-L-Ieucine, 10 min digestion; 0-0, mucosal extract containing 0.74 pg of nitrogen added to 1.5 ,umoles of glycyl-r-valine, 15 min digestion.

each other, however, and were found to be 7.4 for L-alanyl-L-glutamic acid dipeptidase, 7.9 for glycylglycine dipeptidase (EC 3+3.1) and glycyl-L-leucine dipeptidase (EC 3+3.2) and 7.6 for glycyl-L-valine dipeptidase. Lnfluence of metal ions Since it was well known that the activity of dipeptidases is greatly influenced by the presence of different metal ions, the four different dipeptidase systems were studied in this respect, As the assay procedure had been found to be very suitable in studying the activities without addition of metal ions, it was to be expected that even minor changes in the activity owing to inhibition or activation by specific metal ions could be easily studied. However, since it has been found-v that the interaction between metal ions and certain dipeptides can result in the formation of an ultraBiochim, Biophvs, Acta, 105 (X96S) I49-161

L. JOSEFSSON, T . LINDBERG

violet-absorbing complex, special care was taken to study this effect. Such complexes were found to occur between C02+ ions and three of the dipeptides, namely glycylglycine, glycyl-t-Ieucine and glycyl-r.-valine , and the amount of complex formed in creased with the amou nt of metal ion added. The Co2+-gly cylglycine system displayed a particularly high ultraviolet absorption. As ab sorption interfered wit h the assay pro cedu re, the formation of the complex was studied by varying the way of addi ng the metal ions. It was found that if the met al ions were first dissolved ill the buffer solution and then mixed carefully with the subst rate before the addition of the enzyme, the assay was undisturbed in the concentration studied. The presence of C02+ ions was observed not to change th e absorpt ion of the amino acid mixtures. Th erefore, when assaying the influence of the metal ions th e buffer soluti on was made up in serial solutions containing increasing concentrations of the ion from I to IOO f'M. The suitability of this procedure for studying the specificity of metal ions on the different dipeptidase activities is demonstrated in Table I, where the results are given as change in the absorbancy per min and per mg nitrogen present in the enzyme solution. The different dipeptidase activities were measured at their pH optima and in other resp ects according to the st andard procedure given in the E XPERIMENTAL section . F rom th e results obtained it was appar ent that the met al-ion specificity could serve t o different iate the four part icular dip eptidase activities. TA BLE I E FFE CT OF BIV AL E NT METAL IONS ON TH E D I PEPTID AS E ACTIVITI ES OF PIC I NTESTI N AL MUC OSA

Standard as say procedure, op t im al pH. M etal i on add ed

Con ce1l-

iration [mumoles)

°0 .1 I 5 10 0 0.1 I

5 IQ

Mg' +

0 0 .1 I 5 10 0 0.1 I

5 10

LlA m Jm inJm g N L-A l any lt.-glutam ic acid , p hosp hate buffer 19. 0 1 4 ·7 6 .9 6 4. 2 2 4 ·35

Ig ,o

Gly cyl- t.leuc ine , borate buffer

Glycyl-uvaline, borate buffer

0.62 0·72 1.5 1 2.26 2.29

4 0. 6 3 8. 0 35 ·3 2 5. 6

2 7 .0 26.1 26·7 2 5. 0 22 ·7

375

Glyeyl-

glycine , borate buffer

ro .r

0 ,62 0.83 1.20 1. 2 5 I ,s:Z

2 4 ·5 2 4 .6 2 4 ·4 24 · 7 24 ·7

0 .6 2 0 .7 2 0 .7 2 0·73 0.64

2 4· 5 24 .1 2 3 ·7 24. 8 25 .0

0 .62 0 ·59 0·5 4 0 .2 8 0 .0 6 6

18 ,2 1 6 ,7 13 ·2

B iochim : Biop liys, Acta, IOj ( 1 965 ) 149 - 16 1

20 ·4

34·5 26·4 20·3 10 . 1 3 6 .2 3 6 .2 3 7.1

37 · z

~ 3 ·8 2 3 .8 24 .8 20. 2 1 8. 2

2 2 ·4 22 · 3 2 3·3 2 1.8

3 6 .9

z I.8

37 · 5 3 6 .9 31. 7 22 .6 19. 2

23 · 4

z2.6 1 6. 0 9· 3 9. 1

INTESTINAL DlPEPTIDASES. I.

157

This was even more clear when it was found that the anion used in the buffer in the presence of Zn 2+ ions also had a significant influence on the activities (Table II) . This effect was somewhat surpris ing since none of the other metal ions behaved in this way nor was there any effect of the anion in the absence of Zn 2+ ions. L-Alanyl-L-glutamic acid . Co2+ ions showed a potent inhibitory effect resulting in an 80% decrease in activity in the m aximum concentration studied . Mn2+ ions TABLE II EFFECT OF Zn2+ IONS ON THE DIPEPTIDASE ACTI VITIES OF PI G INTESTINAL MUCOSA WITH DIFFERENT BU F F E R SYSTEMS

Standard assay procedure, op t imal pH.

Concentration of Zn H ions added (ml,moles)

LJA220/minlmg N L-A lanY!-Lgluta mic acid D.rM borate buffer

o.i M phosphate

Glycylglycin e o.i M borate buffer

12·3 10·9

I

I 2 .0

5 10

6.21 3. 89

12·f 12·4 12·9 12·9 H .8

Gly cy l-u-ualine

0 .1 lVI borate buffer

o.I M borate buffer

buffer

buffer

0 0. 1

D.r M

phosphat»

Glycyl-u-leucine

0.6 2 0·59 0·54 0.28 0.066

0.88 0.88 0 .81 0·74 0.61

34·5 33·4 34·5 21.2

15·7

0.I1'v1

pic os-

pilate buffer 3f ·5 39·0 37. 6 4°·0 4°·1

0.1 M phos-

pluue buffer

:!3·2 21,5 17. 1 13·3 10·5

23·2 23·2 23·5 23·6 22·7

also red uced the activit y bu t in a more gradual way to a value about 50% of the original activity. Mg2+ ions were found to be without any influence. Zn2+ ions behaved differen tl y depending on the anion present. They were without effect in the phosphate buffer system but with borate buffer, they resulted in nearly 70% inhibition. Glycylglycine. As expected from earlier studies, Co2+ ions were found to be a strong activator, increasing th e activity about four-fold in the higher concentrations studied. Addition of Mn2+ ions resulted in a two-fold activation. Mg2+ ions were without effect. In contrast to the findings with the other clipeptides, in this case Zn2+ ions caused inhibition with both phosph ate and borate buffer. The degree of inhibition was markedly different, however, amounting t o 30% and 9°%, respect ively. Glycyl-L-leucine. Co2+ ions reduce d the activity to about 50% when added in an amount of 10 mzzmoles to th is assay syste m . Mn2t ions had a great er effect, however, and when add ed in the same concentration reduced the activity to about onefourth of the original. As found for the other system s the addition of Mg2+ ions had no influence on the activity while Zn2+ ions contrary to th e other systems studied, affected the activity in both directions dependent on the buffer used. Thus it was found that they increased the activity by about 15% when added with phosphate buffer but decreased the activity more than 50% when added in the borate buffer syst em. Gtycyl-i-oaline. The metal ions were found in genera l to affect this system to a much less extent than the oth ers. C0 2+ ions and Mn2t ions caused only a small B iochim , Biophys. A eta, 105 (1965) 149-161

158

L. JOSEFSSON, T. LINDBERG

reduction in the activity and the Mg 2+ ions, in accordance with the other dipeptidase systems had no effect. Zn 2 + ions had no influence when added with phosphate buffer whilst they resulted in a somewhat more than 50% inhibition when studied in the borate buffer system. Infl~tence

of variations oj the standard assay procedure The effect of changing the relative amounts of the reagents was investigated by measuring the absorbancy at zero time and varying the concentration of one of

TABLE III EFFECT OF VARIATIONS IN WATER VOLUME UPON THE ABSORBANCY AT ZERO TIME

Substrate: glycyl-r.-leucine: enzyme: pig intestinal mucosa extract containing 60.5 fig N Iml. Water added (fll)

14° 15° 160 17°

190 210 220

o.t M borate buffer (PH 7·9) 93 6 869

924

9 16 903 894 85 8

o.x M phosphate buffer (PH 7·9) 783

808 800

771 801

77 2

778

the reagents both above and below the concentration used in the standard procedure. Variations in the amount of enzyme, amount of water, concentration of buffer salt and amount of ethanol added for the precipitation were studied. The results indicated that changes in the enzyme concentration, expressed as amount of total nitrogen added, had no influence on the zero time absorbancy within TABLE IV EFFECT OF VARIATIONS IN BUFFER CONCENTRATION ON 'IHE ABSORBANCY AT ZERO TIME

Substrate: glycyl-z-Ieucine: enzyme: pig intestinal mucosa extract containing 60.5 /-lg N/ml. Concentration of buffer salt (M)

Boraie buffer (PH 7·9)

Phosphate buffer (PH 7.9)

0.033 0. 06 7

0.080 0.100 0.II3

0.133 0. 16 7

Biochim, BioPhys. Acta, 105 (1965) 149-161

INTESTINAL DIPEPTIDASES. 1.

r59

the levels used in the different assays (ef. Influence of enzyme concentration). Similarly, variations in the amount of water in either buffer system were without effect when varied within a range of ± 20% of that used in the standard assay procedure (Table III). The nature and the concentration of buffer salt were, however, found to influence the peptide bond absorbancy within certain limits (Table IV). With the borate buffer system, the absorbancy decreased gradually with increasing concentrations of the salt, while a small increase in the absorbancy was observed with the phosphate buffer at concentrations higher or lower than those used in the standard assay procedure. In the range studied (± 70% variation) the effect on the zero time TABLE V EFFECT OF VA.IUATIONS IN ETHANOL VOLUME UPON THE

ABSORBANCY AT ZERO TIME

Substrate: glycyl-L-leucine; enzyme: pig intestinal mucosa extract containing 60.5 p.g N 1m!. Ethanol added (ml)

o.i M phosphate buffer (PH 7·9)

1.0

80 5 837

1.1

1.2

787

1.3

778

1.4

759 73 8 7 26

1.5 1.6

absorbancy was, however, only about ± 10%. In accordance with expectations an increase in the volume of the precipitating reagent decreased the value for the zero time absorbancy but the change was small in comparison with the variations of the volume of ethanol as shown in Table V. DISCUSSION

Considering first the assay procedure, the many advantages of utilizing the optical difference between dipepticles and their corresponding amino acids for the peptidase activity measurements are apparent. Earlier attempts have been made to utilize the convenience of such a direct spectrophotometric assay procedures." as the titrimetric and colorimetric procedures for determination of dipeptidase activity l O,l1 are inadequate in many respects. These early attempts met, however, with little success, because of the absorbance by extraneous substances present in the test. The introduction of a precipitating reagent to eliminate these substances allows the many advantages of the spectrophotometric analysis to be fully utilized. It furthermore permits dipeptidase activity to be assayed in crude tissue extracts. The accuracy of the method is about ± r%. In addition to the simplicity, high accuracy and low substrate requirement the method is much more sensitive than the hitherto commonly used procedures. The amount of dipeptidase measurable depends on the time of reaction chosen. The ability to measure with certainty 10-4-10-5 mmole of dipepBiochim, Biophys. Acta., 105 (19 65) 149-16 1

160

L.

JOSEFSSON,

T.

LINDBERG

tide hydrolyzed, in combination with the stable conditions of the procedure, permit determinations of dipeptidases of the same small order of magnitude as the dilatometric procedure of LINDERSTROM-LANG AND LANZ I 5• The high sensitivity of the method permits its utilization in analysis of dipeptidase activity in tissue extracts without addition of extra metal ions. On the other hand, it also makes the assay procedure especially suitable for investigations of the effect of metal ions on the dipeptidase activity even in those cases when ultraviolet-absorbing complexes are found in the digest mixtures, e.g. glycylglycine dipeptidase. The small influence of variations in the reagent solutions makes the procedure suitable for routine analysis and allows a wide application in the analysis of most kind of peptidases. However, the high absorbancy by the aromatic amino acids at the wavelength used for measurement imposes certain limitations on the enzyme systems which can be studied by this procedure. Although not many informations on the properties of dipeptidases from intestinal mucosa are available from earlier studies, some considerations relating to our findings may be mentioned. ROBINSON AND SHAW I 6 have reported a double pH optimum for the glycylglycine dipeptidase and for the glycyl-L-Ieucine dipeptidase of the rat intestine. However, studies of peptidases of other tissues splitting those dipeptides have given pH curves with only one maximum l 4 ,17 and we were able to find-" only one pH optimum when extending our studies to include the rat intestinal mucosa. On the other hand the metal-ion specificity demonstrated the dual characteristics earlier frequently found for the dipeptidases. Thus the glycylglycinesplitting activity previously have been found H ,16, 17 to be activated by C02 + and Mn2+ ions in accordance with our findings. Likewise the dual inhibition by C02+ and Mn 2 + ions we observed for the glycyl-L-leucine dipeptidase is in conformity with previous studies 16 ,1 9 . The slight decrease of the activity reported for the glycyl-tleucine-splitting activity when measured in the presence of Mg2+ ions was, however, not observed in our studies. Neither were the other activities influenced by the addition of these ions. The different effect observed for the Zn 2 + ions when added with borate as opposed to phosphate buffer is somewhat surprising because none of the other ions behaved in this manner. Similar findings have, however, been reported by SMITH 19 in investigations on the glycyl-L-leucine dipeptidase from human uterus. The different behaviour may well be interpreted by an ability of phosphate to bind the Zn 2 + ions and thereby eliminate their inhibitory effect. The presence of a lag period in the metal ion interaction frequently reported for the metal-activated enzymes could not be detected in our studies with either the residual systems or the metal ion systems. However, ROSENBERG 2o has cast doubt on the significance of the lag period in his study on carnosinase because he observed that it disappeared with the purification of the enzyme. He suggested that it was an effect of a slow rearrangement of partially denaturated enzyme. Another suggestion is that the lag period may be due to an interaction between the metal ions and substances in the crude enzyme preparation which are not specific for the digestion. This suggestion is supported by the observations-t.w that the lag period diminished when metal ions and enzyme solution were preincubated. The high sensitivity of the present assay procedure allowing the addition of micro-amounts of enzyme solution may allow further light to be shed on this question. The different properties obtained for the four dipeptidase activities studied Biochim, Biophys, Acta, 105 (1965) 149-161

I NT ESTINAL DIPEPTlD ASES . 1.

161

suggest that t hey ar e relate d to four dist inct enzymes. This is in conformit y with general concepts regarding the specificity of the dipeptidases. However, a final conclusion must wait until more ext ensive purifications of the activities have been made.

ACKNOWLEDGEMENTS

Miss G . A . A NDERSSON , Miss K. AN GARDE and Miss 1. G ORANSSON receive our thanks for their skilful techni cal assistanc e. Th e investigation was aided by grants from the Swedish Medical Research Council and the Swedish Nutrition Foundation. REFERENCES I T. K. SH:-lITKA, Federa tion Pro c., 19 (1960) 897. 2 1. DAWSON AND J . PRYSE-DAVI ES, Gastroenterology, 44 (1963) 745. 3 A. C. FRAZER, Advan , Clin . Chem ., 5 (1962) 69. 'I A. DAHLQV1ST, J . B. I-[AMM OND, R. K . CRANE, J. D UNPHY AND A . LITTMAN, Gastroenterology, 45 (19 6 3) 4 88. 5 E. L. SMITH, in P . D . B OYER, H. L ARDY AND K. l\1YRIlACI< , Th e E nzy mes, Vol. 4, Academ ic Press , New York, 1960, p . 1. 6 E. L . SMITH, in S . P . COLQWICK AND N . O. K APLAN , Me thods in Enzymology, Vol. 2, Acad emic Press, New York, 1955, p . 93 . 7 L. J . SAIDEL, A. R. G OLDFARB AND S . WALDMANN, J. Bioi. Chem., 197 (1952) 28 5. 8 F. BIN KLEY AND C. TORRES, A rch. B iochem, B iop hys ., 56 (1960 ) 20 1. 9 A. S CHMITT AND G. SIEBERT , B iochem. Z ., 334 (1961) 9 6 . 10 S. MOORE AND W . H. STEIN, j. B iol. Chem., 2II (1954 ) g 0 7· II W. GRASSMANN AND W. HEYD E, Z. Pliys. Chem., 183 (1929 ) 3 2 . 12 L. JOSEFSSON, N ature, 204 (1964) 78 3. 13 M. P. TO ~1BS , F. S OUTER AND N . F . M ACLABAN, B iochem. J., 73 (195 9) 16 7. i4 E . L . SMITH, J. B ioi. Chem., 17 3 (1948) 57 1. 15 K. LI NDERSTR0M-L ANG AND H. L ANZ, J R., Comp t. R en d. T rau. L ab. Carlsberg, Ser , Cbi m ., 2 1 (193 8) 3 15. 16 G. B . R OBINSON AND B. SHAW, B iochem, j., 77 (19 60) 35 1. 17 H . G. WILCOX AND M. FRIED, B iochem , ] .,87 (19 63) 192. 18 T . LI NDBERG AND L . J OSEFSSON, t o b e published. [ 9 E . L. S MITH, ]. B ioi. Chem., 176 (1948) g. 20 A. R OSENBERG, T he Role of lYI eial Jons in the Catalytic A ction of Peptidases, Almqv is t and Wik sell, Uppsala , 1960 , p . 14 . :2 I W . RADEMAKER AND J . B. J. S OONS, Biochim. Biopby s. A cta, 24 (1957) 209.

B iochim , Biophys . Ac ta, 105 (19 65 ) 149-161