Fractionation of peptidase and esterase activities of human cerebrospinal fluid

136

I~RAIN RESEARCH

FRACTIONATION OF PEPTIDASE AND ESTERASE ACTIVITIES OF H U M A N CEREBROSPINAL FLUID

P. J. RIEKKINEN ANDU. g. RINNE Brain ResearchLaboratory of the Department of Anatomy and the Department of Neurology, University of Turku, Turku (Finland)

(Accepted December 28th, 1967)

INTRODUCTION Human cerebrospinal fluid is known to have both peptidase and esterase activities1,13. Cerebrospinal fluid has both esterase- and tipase-type enzymes! z. Peptidase activity has also been established in cerebrospinal fluid by many authors 3. Both these groups of enzymes have been employed in clinical studies and some correlation has been sought between the activities observed and various diseasesL Experiments need to be made on test animals to correlate the esterase and peptidase activities of cerebrospinal fluid and brain tissue under different exPerimental conditions. Results of such experiments, however, have not been found in the literature. A more detailed study of the character of enzymes in brain and cerebrospinal fluid should be a requisite to such analysis. The purpose of the present work was to fractionate the peptidase and esterase activities of cerebrospinal fluid and to characterize their principal features with the use of chromogenic substrates. MATERIALAND METHODS Cerebrospinalfluid. Cerebrospinal fluid was collected from 16 volunteer subjects with no known neurological disease. The subjects were between 25 and 40 years old. The cerebrospinal fluids were centrifuged and checked for the presence of cells. They were concentrated by freeze-drying to approximately 4 ~ protein content. Substrates. L-Leucyl-fl-naphthylamide, L-arginyl-fl-naphthylamidei L-histidylfl-naphthylamide, L-threonyl-fl-naphthylamide, a-naphthylacetate, naphthol-ASacetate, a-naphthylbutyrate, fl-naphthyllaurate and acetylthiochotine iodide were commercial preparations obtained from Sigma Chemical Company (Ohio, U.S.A 3. Stock solutions were prepared of each substrate at a concentration of 1 mM by dissolving the substrate in methanol and adding buffer so that the final concentration of methanol was 4 0 ~ with all substrates except /%naphthyllaurate, with which methanol at 70 ~ was used. Enzyme assays. Hydrolysis rates of the substrates were measured in an inBrain Research, 9 (1968) 136-144

LIQUOR ESTERASES AND PEPTIDASES

137

cubation medium made up as follows: 0.1 M Michael's buffer 1.0 ml; enzyme fraction 0.5 ml; substrate stock solution 0.5 ml. Incubation was carried out at a constant temperature (37°C) for 10 min to several hours depending on the substrate used. When naphthyl substrates were used, the liberated naphthol was coupled with diazonium salt by adding 0.5 ml aqueous Fast Garnett GBC solution (Edward Gurr, London, U.K.) (2 mg/ml) in which l0 Tween 20 was added to disperse the coloured azo dye product. Two minutes later 1.0 ml 1 M acetate buffer, pH 4.2, was added to stop the enzymic reaction. Colour intensities at 540 m# were measured 10 min later in a Beckman DB spectrophotometer. Naphthylamines liberated during incubation were determined as follows: reaction was stopped by adding 1.0 ml 1 M acetate in which 1 0 ~ Tween 20 was used to disperse the dye product (concentration of Fast Garnett GBC 1.0 mg/ml), and, after 15 min, the colour intensity was measured at 525 m# in the spectrophotometer. Standard curves were prepared from alcoholic solutions of the corresponding naphthol derivative and 2-naphthylamine (Merck, Darmstadt, Germany) under identical conditions. Affectors. The following affectors were used to characterize the separate activities. E-600 (diethyl-p-nitrophenylphosphate, Bayer, Germany), DFP (diisopropylfluorophosphate, Sigma Chemical Company, Ohio, U.S.A.), eserine sulphate (Nutritional Biochemicals Corporation, Ohio, U.S.A.), p-chloromercuribenzoate (PCMB), sodium taurocholate, cysteine-HCl, monoiodoacetic acid and the following inorganic salts at a concentration of 2-5 mM: calcium chloride, cupric chloride, mercuric chloride, magnesium chloride, potassium cyanide and lead nitrate. Stock solutions were prepared from all affectors in buffer solutions at a concentration 4 times that required in the final incubation medium. In studies with affectors, 0.5 ml of buffer in the incubation medium was replaced by buffer containing the affector. Chromatography. Diethylaminoethylcellulose (DEAE-cellulose, Whatman, Floc DE-50) and carboxymethylcellulose (CM-cellulose) were obtained from Balston (U.K.). Sephadex G-100 and G-200 (particle size 10-40) were commercial preparations from Pharmacia, Uppsala (Sweden) and were handled according to the manufacturer's directions. Before running, packed columns were equilibrated with starting buffer. Starch-gel electrophoresis. The hydrolysed starch was obtained from Connaught Medical Research Laboratories, Toronto (Canada), and the gels were prepared in plastic trays (20 c m x 8 cm × 0.5 cm), according to the manufacturer's directions. Protein bands in the gels were made visible with Amido Black. A strip was cut from a gel, put into a solution containing different substrates mentioned earlier and also Fast Garnett GBC (1.0 mg/ml) and Tween 20 (10 ~o). Colour bands which formed in the gel indicated the location of different enzyme components. Molecular weight determination. The approximate molecular weight of the enzyme was determined with a Sephadex G-100 column (2 cm × 150 cm) in which 10 mg bovine serum albumin, bovine ribonuclease and 200 #g trypsin were applied together with 1.0 nag of each final preparation. A small crystal of blue dextran was applied at the same time in order to check the column. The elution was carried out with 10 m M Tris-HCl buffer (pH 7.0) containing 0.1 M NaCl. The elution rate was

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P.J.

R I E K K I N E N A N D U. K. R I N N E

about 5 ml/15 min and the fractional volume was 5.0 ml. Albumin was identified as protein, ribonuclease also as protein, and trypsin was demonstrated with N-a-benzoylDL-arginine p-nitroanilide. The activity of the applied enzyme preparation was demonstrated with its substrate in the usual way. A Whitaker's plot was made to show the distribution of proteins and a standard value of 70,000 was used for albumin, 23,800 for trypsin and 12,000 for ribonuclease. Proteins and enzymes. Bovine serum albumin, twice crystallized salt-free trypsin and bovine ribonuclease were preparations obtained from Nutritional Biochemicals Corporation, Ohio (U.S.A.). For other methodological details, we refer the reader to our earlier reports s,°. RESULTS

D EAE-cellulose chromatography Fig. 1 shows a typical distribution of peptidase activity in a DEAE,cellulose chromatography column. Leucyl-~-naphthylamide yielded three separate peptidase DEAE-

ceLLuLose

- -

chromatography

L-teucyt~~ I.-arginyt--~

-nmphthytamlde naphthytamide

-

0,6

.o.s

g

F

o

IJ 0.;

~.~~ 10

20

3'0

k0

50 60 7Q Fraction ~ u m b e r

80

do

lbo

11o 12o

Fig. 1. Distribution of peptidase activity in DEAE-cellulose chromatography. Continuous linear gradient from 20 mM to 0.5 M NaC1 in 20 mM Tris-HCI buffer, 7.0 ml concentrated solution containing 294 nag protein was applied to the column. Column 3 cm ~ 40 cm. hydrostatic pressure 45 cm H20, flow rate 0.6 ml/min, fraction volume 5.0 ml. Incubation time 20 rain for leucyl- and 50 rain for arginyl-2-naphthylamide. In further chromatography, fractions 6-15 were pooled for peak I, fractions 26-36 for peak II and fractions 50--60 for peak III. components. The first of these components was eluted almost immediately from the D E A E column at p H 7.0. When different naphthylamide substrates were tested, partial separation was established, but at the second peak both leucyl- and arginylfl-naphthylamidase activity was demonstrated. The properties of this peak appear to argue in favour of aminopeptidase B. An attempt was made to separate these two possible components on Sephadex and CM-cellulose. N o chromatographic separation occurred in the Sephadex G-200 column, whereas in CM-ceUulose the peaks differed somewhat (Fig. 3), which supports the assumption that the activity was produced by two or possibly several enzyme components. The third peptidase component Brain Research, 9 (1968) 136-144

139

LIQUOR ESTERASES AND PEPTIDASES

DEAE-ceU_utose chromatography O'61

-- ~-naphthyL-acetate .... acetyLthiochofineiodide ,~-naphthyL-taurate . . . . . . . . . . .

~

.~

I

o.sg

0.1

~

t

20

z,O

60

Fraction number

Fig. 2. Distribution following incubation iodide and 1 h for further fractionation, peak III.

80

100

~

120

of esterase activities in DEAE-cellulose. For the different substrates the times were used: 10 min for a-naphthylacetate, 20 min for acetylthiocholine /3-naphthyllaurate. Other experimental conditions were as for Fig. 1. In fractions 10-18 were pooled, peak I; fractions 35-45, peak II; fractions 63-76,

coincided fairly extensively with the proteinase component, but differed quite considerably from the esterases. Of the esterase substrates tested, naphthol-AS-acetate, a-naphthylacetate, a-naphthylbutyrate, a-naphthyllaurate and acetylthiocholine iodide, each gave one or more activities in DEAE chromatography, as can be seen from Fig. 2. a-Naphthylacetate and -butyrate and naphthol-AS-acetate were hydrolysed at the first esterase peak in DEAE-cellulose, but acetylthiocholine iodide and flnaphthyllaurate were not. In contrast, pronounced hydrolysis of acetylthiocholine iodide and slight hydrolysis of a-naphthylacetate were observed at the second esterase peak, which coincided with a proteinase component. Slight hydrolysis of all the naphthyl esters tested was observed at the third esterase peak, but it was characteristic of this peak that it also displayed hydrolysis of lipase substrate, suggesting that this component is the source of the lipase activity of cerebrospinal fluid. In addition,

CH-ceLLuLose chromatography ........

L- T.eucyl.-,~-naphthytamide L - a r g i n y L - ~ - naphthyLamide

0.6

~o L

0.3

o:

o

o.1 ," ,

,

20

,

,

,

40

,

,

60 Fraction

.

,

8O

140

120

number

Fig. 3. Distribution of peptidases in CM-cellulose chromatography. Continuous linear gradient from 0 to 0.5 M NaC1 in 20 m M Tris-HC1 buffer at pH 7.0 was used. 5.0 ml concentrated solution containing 210 mg protein was applied to the column. The column was 3 cm × 40 cm, hydrostatic pressure 50 cm H20, flow rate 0.8 ml/min, fraction volume 5.0 ml. Incubation time 20 min for leucyl- and 50 min for arginyl-2-naphthylamide.

Brain Research, 9 (1968) 136-144

140

p.J. RIEKKINEN AND U. K. RINNE

slight a-naphthylacetate, naphthol-AS-acetate and a-naphthylbutyrate bands were observed near the last-mentioned peptidase component.

Sephadex G-200 chromatography Only two peptidase activities and, slightly separated from them, three esterase activities, were elicited in Sephadex G-200 c h r o m a t o g r a p h y . A poorer resolution was obtained with Sephadex than with D E A E c h r o m a t o g r a p h y : the peaks obtained with Sephadex were not characterized at all, and Sephadex G-200 was only used for purification o f the second phase when the peaks obtained from D E A E were fractionated further.

Further purification by Sephadex G-200 chromatography of activities elicited from DEAE Each peptidase c o m p o n e n t was run separately t h r o u g h the Sephadex G-200 column, and protein, as well as peptidase and esterase activities were determined for Sephadex G - 2 0 0 chromatography

. . . . ol.teu cyt-.8-naphthyta mide ........ d--it ginyt--,t$-n~ytami de

0.5.

- -

~-naphthyt-

acetate

i ,< 0.2

2'0

~0

60 Fraction

8O

100

number

Fig. 4. Distribution of peptidase and esterase activity in Sephadex G-200 chromatography. Eluting solution 0.1 M NaCI in 20 mM Tris-HCl buffer, pH 7.0. Concentrated solution (6 ml containing 252 mg protein) was applied to the column. Column 4 cm × 80 cm. fraction volume 10.0 ml, and number of fractions 100. Incubation times were the same as those in Fig. 1 for corresponding substrates. Hydrostatic pressure 30 cm H20, flow rate 0.7 ml/min.

each run. Partially purified peptidase peaks 1, 2 and 3 a n d also three esterase activities were elicited. One of the esterase components was choline esterase and one was lipase. However. as in the D E A E stage, complete separation of the first peptidase c o m p o n e n t and the first esterase c o m p o n e n t obtained f r o m D E A E was not achieved even in Sephadex gel filtration. O n the other hand, the resolution o f other peaks was fairly successful. Table I shows the substrate specificity o f the peptidase activities obtained. Table II shows the p H optima and molecular weights. Each peptidase peak obviously

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LIQUOR ESTERASESAND PEPTIDASES TABLE I SUBSTRATE SPECIFICITY OF PARTIALLY PURIFIED PEPTIDASES

Hydrolytic activity (m/~moles/mg prot./min)

Substrate

L-Leucyl-/%naphthylamide L-Arginyl-fl-naphthylamide L-Alanyl-fi-naphthylamide L-Methionyl-/3-naphthylamide L-Glycyl-/4-naphthylamide L-Histidyl-/%naphthylamide L-Threonyl-/3-naphthylamide L-Seryl-fl-naphthylamide

Peak 1

Peak2

Peak3

45 1! 17 2 13 0 II 4

32 41 19 4 11 0 5 28

72 8 6 1 12 0 24 13

Peak 1

Peak 2

Peak 3

7.4 80,000

6.8 92,000

7.0 140,000

TABLE 11 MOLECULAR WEIGHTS A N D

pH

pH optimum Molecular weight

OPTIMA OF DIFFERENT PEPTIDASES

contained an enzyme group of its own type. The peaks were evidently not yet pure. At pH 7.4 the first component hydrolysed leucyl-fl-naphthylamide most rapidly, then alanyl- slowly, also valyl- and methionyl-, but it hydrolysed arginyl- more slowly than leucyl-fl-naphthylamides. The second component hydrolysed both arginyl- and leucyl-fl-naphthylamides and obviously, therefore, contained at least two different enzyme components. The pH optimum was 6.8. The third component hydrolysed leucyl-fl-naphthylamide most rapidly, but also hydrolysed other naphthylamides, though slowly, including even arginylnaphthylamide. The molecular weight of the middle peak was 92,000, that of the first 80,000 and that of the last about 140,000 according to the Whitaker's plot. The first two esterase components were fairly unspecific and obviously belonged to the group of so-called fl-esterases. Choline esterase typing, as appears from Table III, displayed characteristics of distinctly non-specific choline esterase. The lipase component was activated by both calcium chloride and taurocholate, and it chiefly hydrolysed the naphthyl esters of fatty acids with a longer chain, thus arguing for the lipase character of the enzymes. Starch-gel electrophoresis

Fig. 5 shows that leucyl-naphthylamide, in Poulik's buffer system gave three different bands, and that one of them, the first, became visible also when arginylBrain Research, 9 (1968) 136-144

142

i,. j . R I E K K I N I : N A N D ~[ K. R I N N [

TABLE Ill AFFECTOR CHARACTERISTICS OF ESTERASE COMPONENTS

Affector

CaCI2 CuCI,2 MgCle HgCI DFP E-600 Eserine KCN PCMB Iodoacetic acid Sodium taurocholate

Concentration

Percentage change in activitv

(raM)

Peak I

Peak 2

Peak 3

l0 I l0 I 0.01 0.01 0.01 I I 1 0. I

0 - 36 0 40 100 100 60 40 0 0 0

0 - 15 0 - 70 100 100 t00 l O0 0 0 0

35 0 22 0 0 0 t) l0 0 t) 42

m

II

I

l

I L,u-.O-NA

_ _ _ _ _ 1 Arg-~-NA

II

]l l

l

l

l

Il

II

|

l

[11

l

II

II t Naphthyt acetate ] Napthot-AS ac,t*-te I

I Naphthyt butyrate

Fig. 5. Fractionation of peptidases and esterases by starch-gel electrophoresis in continuous Poulik's buffer system. Running time 6 h. About 0.5 ml concentrated: solUtion was applied~t: the origin. After running, gel strips were sliced and activity bands were made visible as presented in the section on Materials and Methods. was used as substrate. O n the other h a n d , six fairly distinct b a n d s were m a d e visible with a-naphthylacetate, a n d two of them occurred at the same sites as the b a n d s o b t a i n e d with the leucyl c o m p o u n d . The results of the starch-gel electrophoresis indicate that the resolution m e t h o d s using c o l u m n c h r o m a t o g r a p h y were n o t able u n d e r these experimental c o n d i t i o n s effectively to separate the esterase c o m p o n e n t s . DISCUSSION

The results obtained show that h u m a n cerebrospinal fluid c o n t a i n s several different types of peptidase and esterase. A t least three different peptidases, two non-specific esterases, a non-specific choline esterase a n d a lipase were distinguished, a n d still more esterase c o m p o n e n t s were detected with the aid of starch-gel electrophoresis. In the characterization of the enzymes the peptidases a n d esterases corBrain Research, 9 (1968) 136-144

143

LIQUOR ESTERASES AND PEPTIDASES

respond well with the isoenzymes described by other authors v. Contrary to the results obtained by many other workers 5, we were not able to distinguish between the arginyland leucyl-/~-naphthylamidase activities of ccrebrospinal fluid. The affector characteristics of the peaks showed certain definite similarities with the aminopeptidase types previously described. In fact, it is possible from these properties to distinguish these different components of cerebrospinal fluid as separate entities. Markedly increased leucyl-/3-naphthylamidase activity in cerebrospinal fluid has been demonstrated in, for example, turnouts 3. However, it is difficult to say in the light of the present results which enzyme component, or possibly components, has increased in the cerebrospinal fluid. This led us to develop fractionation methods and characterization of various isoenzymes as means of demonstrating that these different activities were due to individual entities. If cerebrospinal fluid esterases and peptidases are to be used in clinical determinations, their origin should be known. To this end, a concurrent study should be made, by for example immunological methods, of the peptidase and esterase composition of cerebrospinal fluid and different nerve tissues. The changes that occur during the course of certain diseases should be studied more accurately with the aid of the results thus obtained, i.e. it may be possible to ascertain whence the isoenzymes in question have been released into the cerebrospinal fluid. The enzyme types of cerebrospinal fluid must naturally be determined first. Studies of this kind have indeed been conducted, for example with lactic acid dehydrogenase 6, but not on esterases or peptidases. Different enzymic activities of cerebrospinal fluid have been studied fairly extensively and an endeavour has been made to utilize them clinically 4. We have tried here and in earlier studies to classify various esterases, peptidases and proteinases in the acute and chronic stages of multiple sclerosis, in patients with brain tumours, patients suffering from vascular headache, and in a control seriesl0,1k SUMMARY

Human cerebrospinal fluid was collected, concentrated and fractionated by DEAE and Sephadex chromatography and starch-gel electrophoresis. Three peptidase components were distinguished. Each had its own pH optimum and a different substrate specificity, different affector characteristics and molecular weight. Two esterases and a non-specific choline esterase were distinguished by the chromatographic method and at least six separate esterases were distinguished by starch-gel electrophoresis. One of these components also hydrolysed suhstrates of the lipase type. The affector properties were also tested. It was observed that human cerebrospinal fluid contains more types of peptidases and esterases than earlier results have suggested. The possible secretion of enzymes from the brain and their significance for diagnosis are discussed. ACKNOWLEDGEMENT

This work was aided by a grant from the Sigrid Jus61ius Stiftelse. Brain Research, 9 (1968) 136-144

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r,. j. R|EKKINEN AND f_ K. RINNti

REFERENCES 1 COLLIN, K, G., AND ROSSITER, R. J., Cholinesterase of cerebrospinal fluid, Canad. J. Res., tf, 27 (1949) 327-330. 2 GERHARDT, W., CLAUSEN,E., CHRISTENSEN,E., AND R.HSHEDE,J., Changes of LDH isoeazymes, esterases, acid phosphatases and proteins in malignant and benign human brain tumors, Acta neurol, scand., 39 (1963) 85-90. 3 GREEN, J. P., AND PERRY, M., Leucineaminopeptidase activity in cerebrospinal fluid, Nett,'ology (Minneap.), 13 (1963) 924-926. 4 HEITMANN, R., LOSER, R., UND STAMMLER, A., Beitrfige zur Fermentbestimmung im Liquor cerebrospinalis, Dtsch. Z. Nervenheilk., 186 (1964) 121-136. 5 HoPsu, V. K., M.~KINEN, K. K., AND CLENNER, G. G., Purification of a mammalian peptidase selective for N-terminal arginine and ly sine residues: aminopeptidase, B., Arch. Biochem. BDphys., 114 (1966) 557-566. 6 LINDHOLM, U., VRETHAMMAR,T., AND ,t~BERG, B., Isoenzymes of lactic acid dehydrogenase in brain damage, Nord. Med., 77 (1967) 337-340. 7 PATTERSON,E. K., HStAO, S, H., AND KEPPEL, A., Studies on dipeptidases and anainopeptidases. I. Distinction between leucine-aminopeptidase and enzymes that hydrolyze L-leucyl,beta.naphthytamide, J. biol. Chem., 238 (1963) 3611-3620. 8 RIEKKINEN, P. J., AND HOPSU, V. K., Trypsin-like enzymes in salivary glands, Ann. Med. exp. Fenn., 43 (1965) 6-14. 9 RIEKKINEN,P. J., AND HOPSU, V. K., Studies in salivary glands. Hydrolysis of chromogenic ester substrates, Ann. Med. exp. Fenn., 43 (1965) 15-22. l0 RIEKKINEN, P. J., AND RINNE, U. K,, Proteinases in human cerebrospinal fluid, J. neurol. Sci., 7 (1963) 97-106. 11 RINNE, U. K., AND R1EKKINEN, P. J., Esterase, peptidase, and proteinase activities of humzn cerebrospinal fluid in multiple sclerosis, Acta neurol, scand., (in press). 12 SPlEGEL-AOOLF, M., BAIRD, H., AND KOLUAS, D., Lipases in cerebrospinal fluid in vario:s neurological conditions especially infantile amaurotic idiocy, Confin. neurol. (Basel), 17 (1957) 310-313. 13 WIECHERT,P., Vorkommen und Aktivit~it von Peptidasen im Liquor cerebrospinalis,Acta biol. med. germ., 16 (1966) 11-14.

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