Dipeptidyl-aminopeptidases and aminopeptidases in Dictyostelium discoideum

Vol. 127, No. 3 , 1 9 8 5

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 962-958

Ma~h 2 9 , 1 9 8 5

DIPEPTIDYL-~41NOPEPTIDASES AND AMINOPEPTIDASES IN DICTYOSTELIUH DISCOIDEU~ Samuel A. T. Chan, Karim Toursarkissian, John P. Sweeney and Theodore H. D. Jones Department of Chemistry, University of San Francisco San Francisco, California 94117-1080 Received February 15, 1985

Extracts prepared from culminating cells of D~ctyostelium discoideum have been found to contain dipeptidyl-aminopeptidases I (EC 3.4.14.1), II (EC 3.4.14.2), III (EC 3.4.14.4), arginine aminopeptidase (EC 3.4.11.6) and valine aminopeptidase. Dipeptidyl-aminopeptidase III was the most active of the dipeptidyl-aminopeptidases; its molecular weight was 158,000, with a pH optimum of 10.2 and gave a single peak of activity on gel-filtration or when fractionated by chromatofocusing. The specific activities of dipeptidyl-aminopeptidases I and III increased during development being highest during the culmination stage before decreasing during sorocarp formation; dipeptidyl-aminopeptidase II and arginine aminopeptidase decreased progressively throughout development. The presence of these dipeptidyl-aminopeptidases suggests the possibility that processing of peptides may be necessary during the development of Dictyostelium. © 1985 Academic Press, Inc.

Proteinases and peptidases are critically important enzymes in the metabolism of most organisms being responsible for protein degra 5ation, remodelling of structural proteins, regulation of enzymes, 9rocessing of peptide hormones and for other molecular modifications ~hich control cell growth, differentiation and function

~i).

Re-

:ently interest in understanding protein metabolism in Dictyostelium tas increased because of the prominence of protein degradation in he development of this organism.

Cells of Dictyostelium multiply

s single amoebae feeding on bacteria; when the supply of nutrients s exhausted the amoebae aggregate to form a pseudoplasmodium which itimately develops into a mature sorocarp consisting of dead stalk

~bbreviations used are: DAP, dipeptidyl-aminopeptidase; ~-NA, ~-naphthylamine; AMC, 7-amido-4-methylcoumarin; MES, morpholinothanesulfonic acid 0006-291X/85 $1.50 Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

962

'ol. 127, No. 3, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

[Is and viable spores

(2).

Protein degradation during development

~m amoebae to sorocarp is responsible for the loss of more than 50% the dry weight of the cell and provides the amino acids whose ox~tive catabolism is the major source of energy during developat (2).

Several proteinases active on a variety of protein sub-

rates at acid pH have recently been isolated and characterized (3,4). mplete protein hydrolysis also requires the action of peptidases t, in contrast to the proteinases,

there is relatively little

own about peptidases in Dictyostelium. .s been partially purified

A leucine aminopeptidase

(5) and several other aminopeptidase

:tivities have been detected

(6,7).

Peptidases are of particular

iterest because they are involved in regulating enzyme and peptide ~rmone activity.

Since such regulation might have a significant

~le in the developmental program we have tested extracts of D igtyotelium for the presence of certain peptidases and report here the ccurrence in developing cells of activities corresponding to the nzymes dipeptidyl-aminopeptidases

I, II, and III (DAP I, II and III)

s well as arginine aminopeptidase and valine aminopeptidase. [ATERIALS AND METIIODS Gly-arg-7-amido-4-methylcoumarin hydrochloride was purchased :rom Cambridge Research Biochemicals; all other substrates and Sepha:ryl S-200 were from Sigma Chemical Co. Polybuffer exchanger PBE )4 and polybuffer 74 for chromatofocusing were from Pharmacia Fine ~hemicals. Dictyostelium discoideum NC-4 was grown on sheets of E.coli, larvested at different stages of development and disrupted by one :ycle of freeze-thawing followed by hand-homogenization (9). These :ell-free extracts were centrifuged at 10,000g for i0 min. and the supernatants were dialyzed exhaustively against I0 mM morpholinoethanesulfonic acid (MES) buffer, pH 6.5, and used for all enzyme studies. DAP I, If, III and IV were assayed in reaction mixtures containing buffer, substrate, 2 ~M 2-mercaptoethanol and cell-free extract to give a final volume of 5.0 mL. Buffers and substrates used were: 8 ~q MES pH 6.5 and 200 uM gly-arg-AMC hydrochloride for DAP I; 40 mM MES pH 6.5 and 160 ~M lys-ala-~-NA for DAP II; 62 mM Tris-HCl pH 9.0 and 18 ~M arg-arg-B-NA for DAP III; 62 ~LM Tris-HCl pH 8.0 and 200 ~M gly-pro-~-NA for DAP IV. Aminopeptidases were assayed in reaction mixtures containing 26 mM sodium phosphate pH 7.2 and either 166 ~M arg-~-NA or 166 ~M lys-~-NA or 166 ~M val-~-NA with 200 mM NaCI in a total volume of 4.8 mL. All reactions were incubated at 22°C and the increase in fluorescence was measured either in a Turner Fluorometer Model iii or in a Perkin-Elmer Fluor963

Vol. 127, No. 3, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

escence Spectrophotometer Model 650. For pH studies) the customary buffer was replaced with an appropriate buffer of the same concentration and the pH of the mixture was measured following reaction. Gel filtration chromatography was performed on a Sephacryl S-200 column ~39 x 1.5 cm ID) with a sample size of 0.6 mL, flow rate of 4.5 mL/hr and fraction volume of 3.0 mL. Chromatofocusing was performed over the pH range 8-4 on a column (18 x 0.7 cm ID) of polybuffer exchanger PBE 94 with sample size 0.3 mL, flow rate 5.5 mL/hr and fraction volume of 1.0 mL. RESULTS AND DISCUSSION Cell-free extracts of Dictyostelium harvested at the culmination stage of development showed significant activity in converting argarg-~-NA to ~-NA.

Specific activities for different preparations

ranged from i0 to 50 nmoles

8-NA produced/min/mg protein.

Such acti-

vity could be due either to one enzyme cleaving arg-arg-~-NA to argarg and 8-NA (analogous to DAP III) or to one or more enzymes which cleave the arg-arg-~-NA to arg-~-NA and arg followed by cleavage of the arg-~-NA to ~-NA and arg

(analogous to arginine aminopeptidase).

Extracts incubated with arg-~-NA did show substantial activity at pH 7.2, with typical specific activities of 15-80 nmoles ~-NA proJuced/min/mg protein, but the activity at pH 9.0 was always lower ~ith arg-~-NA than with arg-arg-~-NA.

Thus the extracts must con-

:ain separate aminopeptidase and DAP III activities.

This was con-

~irmed by the observation that the activities towards arg-~-NA and ~rg-arg-~-NA had very different pH profiles and at the pH optimum or cleavage of arg-arg-B-NA, pH 10.2, the extracts showed no actiity at all towards arg-B-NA

(fig. i).

E.coli cultures harvested

n the same way by hand-homogenization showed no activity

(<0.01

moles/min/mg protein) with arg-arg-~-NA or arg-~-NA. Physical separation of the aminopeptidase and DAP III was acieved by gel-filtration chromatography and by chromatofocusing. el-filtration,

On

a single peak of activity was obtained for each en-

yme but these overlapped substantially

(fig. 2). Calibration of the

~lumn with protein standards gave molecular weights for the amino~ptidase and DAP III of 165,000 and 158,000 respectively. 964

Chromato-

Vol. 127, No. 3, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BUFFERS ~. M E S • MOPS o CHES D CAPS

600

~.~

DO

"~

ar,-. -NA\

DO

500 !

*rll'al~" ~ -HA

arg- ~ -HA

400 '

300

00 .~

'00

200 100

00 O ~

I

I

I

0 2 4

6

8-10 12 14 16

I

0

4

5

6

7

8

9

10

11

O

12

.o.~.~.o.eD----

pH

20

24

FRACTION

pH profiles of the enzymes active on the 8-naphthylamides of -arginine, lysyl-arginine and arginine, and on glycyl-arginineAMC. Cell-free extract from the culmination stage was incubated with the substrates in different buffers and the rate of formation of the fluorescent product was measured, pH was measured in the reaction mixtures following incubation. Gel-filtration chromatography of the enzymes active on the hylamide derivatives of arginyl-arginine and of arginine. Cell-free extract from the culmination stage was fractionated on a column of Sephacryl S-200 and the fractions assayed for enzyme activity towards the two substrates.

ocusing

separated

the a m i n o p e p t i d a s e

ach being r e c o v e r e d

as a single peak of a c t i v i t y with pI values

.65 and 5.91 r e s p e c t i v e l y To determine minopeptidases

and DAP III almost completely,

(fig.

3).

whether Dictyostelium

and a m i n o p e p t i d a s e s ,

contains

extracts

'ere tested w i t h a s e l e c t i o n of f l u o r o g e n i c or the d e t e c t i o n

of these two classes

"ities c o r r e s p o n d i n g .ine a m i n o p e p t i d a s e ~Table i). Orig.

i) in contrast

tan tissues

to DAP I, DAP II,

other dipeptidyl-

of c u l m i n a t i n g

substrates

of enzymes.

Significant

to the c o r r e s p o n d i n g

ively). 965

acti-

and va-

no DAP IV a c t i v i t y was found

of the DAP I and DAP II were

(pH optima = 5-6 and 4.5-5.5

cells

commonly used

lysine a m i n o p e p t i d a s e

were also detected;

The pH optima

of

activities

7.3 and 7.4

found in mammal-

for DAP I and II respect-

28

Vol. 127, No. 3, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lO0

a~g-arg~-NA-~ .,~--arg-~-NA 75

8

7 -=

16 pH ~=

sc

5

N

,z,,

4 "-=

t~ val'~'NA

25

0

10

20

30

40

50

60

FRACTION NUMBER

Separation by chromatofocusing of enzymes active on the hylamides of arginyl-arginine, arginine and valine. Cellfree extract from the culmination stage was fractionated on polybuffer exchanger PBE 94 and fractions were tested for production of 8-naphthylamine from the ~-naphthylamide substrates shown. The relative activity towards valine-8-naphthylamide is one-tenth of that shown, pH was measured in the separated fractions. Recovery of enzyme activity from the column was 100% and 113% for the DAP Ill and arginine aminopeptidase respectively.

Table I.

Specific enzyme activities in cell-free extracts of Dictyostelium

SUBSTRATE

SPECIFIC ACTIVITY

ENZ~dE

nmoles/min per mg. protein gly-arg-AMC

0.11

DAP I

lys-arg-8-NA

1.92

DAP II

arg-arg-8-NA

I0.8

DAP III

gly-pro-~-NA

<0.01

DAP IV

arg- B-NA

23.5

arginine aminopeptidase

lys-8-NA

30.5

lysine aminopeptidase

val- ~ -NA

0.63

valine aminopeptidase

Cell-free extract from the culmination stage was assayed with different substrates, as described in MATERIALS AND METHODS, except that DAP III activity was measured at pH 10.2.

966

V;)l. 127, No. 3, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The lysine aminopeptidase and arginine aminopeptidase activities ,ear to be due to a single enzyme since the two activities could be separated by gel-filtration chromatography,

chromatofocusing

ion-exchange chromatography and were always recovered as a single Ik of activity in which the fractions showed a constant ratio, 5, of lysine-B-NA hydrolysis to arginine-B-NA hydrolysis. The valine aminopeptidase could be partially separated from the ginine aminopeptidase by chromatofocusing

(fig. 3). Like the valine

inopeptidase activity of human erythrocytes

(I0) it was stimulated

the presence of chloride ions and was therefore routinely assayed 200 mM NaCI which resulted in 80% stimulation.

The specific acti-

ty of the valine aminopeptidase was typically 3% of the specific tivity of the arginine aminopeptidase. In mammalian tissues, the DAP enzymes are thought to be involv[ in the processing of peptide hormones rather than in general pro~in degradation

~8, ii).

Their occurrence in significant amounts

Dictyostelium suggests the possible occurrence of peptide hor~nes and peptide processing in this microorganism.

If so, one would

cedict that they would appear at specific stages in the development E the microorganism.

To test this possibility,

the specific activi-

ies of the DAP I, II and III and arginine aminopeptidase in Dictyotelium were determined at different stages of growth and development Table 2).

The specific activities of DAP II and arginine aminopep-

idase decreased progressively throughout development but the specifc activities of DAP I and III increased after aggregation, reaching maximum at the culmination stage before decreasing again. The studies described here demonstrate the presence in Dictyo~telium of DAP I, II and III (as well as at least two aminopeptilases) which have been reported to be responsible for the processing ~f peptide hormones in mammalian tissues

(8, II). These enzymes vary

in amount at specific stages during the developmental program and 967

Vol. 127, No. 3, 1985

Table 2.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Enzyme specific activities at different stages of development

DEVELOI~iENTAL STAGE

ENZYME SPECIFIC ACTIVITY nmoles/min per mg. protein DAP I

DAP II

DAP III

arginine aminopeptidase

aggregation

0.Ii

3.0

9.4

14.2

early culmination

0.14

2.2

12.3

14.2

late culmination

0.15

0.81

13.1

9.1

sorocarp formation

0.06

0.24

8.6

4.3

Cells were harvested at different developmental stages, disrupted by homogenization and the supernatants then dialyzed and assayed under standard conditions. DAP III was measured at pH 10.2.

suggest the possibility mediating trolling

cell-cell

that peptide hormones

intercommunication

cell movements

may be involved

in Dictyostelium

in

and in con-

during morphogenesis.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial support of Research Corporation, Tucson, Arizona and the Lily Drake Research Fund of U.S.F. REFERENCES I°

Holzer H. and Heinrich,

P.C.

(1980) Annu. Rev. Biochem.

2.

Loomis, W.F. (1975) Dictyostelium ic Press Inc., New York.

discoideum,

North, M.J.

(1982) Microbiol.Reviews

North, M.J.

and Whyte, A.

Firtel,

Fong, D. and Rutherford, North, M.J.

C.L.

(1982) Exp.Mycol.

R.W.

63-91.

pp. 88-90, Academ-

46, 308-342.

(1984) J.Gen.Micro.

R.A. and Brackenbury,

49,

130,

123-134.

(1972) Dev. Biol.

(1978) J.Bacteriol.

27, 307-321.

134,

521-527.

6, 345-362.

McDonald, J.K. and Schwabe, C. (1977) Proteinases in Mammalian Cells and Tissues, pp 314-376, North-Holland Pub. Co., Amsterdam.

9,

Ward,

C. and Wright,

B.E.

(1965) Biochemistry

S~derling E., Hujanen E. and M~kinen, Medicine 26, 231-238. t.

Lee, C. and Snyder,

S.H.

K.K.

(1981)Biochemical

(1982) J.Biol. Chem. 968

4, 2021-2027.

257, 12043-12050.