Purification and properties of human acid-thermostable ribonucleases, and diagnosis of childhood pancreatic fibrosis

231

Clinica Chimica Acta, 67 (1976) 231-243 @ Elsevier Scientific Publishing Company,

Amsterdam

- Printed

in The Netherlands

CCA 7515

ALICJA

BARDOi,

HALINA

SIERAKOWSKA

and DAVID

SHUGAR

National Research Institute of Mother and Child, 01-211 Warszawa, and Institute of Biochemistry and Biophysics, Academy of Sciences, 02-532 Warszawa (Poland) (Received

August

16, 1975)

Summary Acid-thermostable ribonucleases were isolated from human pancreas, duodenal contents, liver, spleen, serum and urine, and purified 15-lOOO-fold. The pH optima, ionic requirements, and some of the specificity requirements, of these enzymes were investigated. The isolated enzymes formed two distinct groups: (a) The ribonucleases of the pancreas, duodenal contents and fraction A of serum and urine exhibit a pH optimum of 8.5, are inhibited by Zn” and Cu’+, and relatively rapidly hydrolyze the synthetic substrate uridine 3’-(a-naphthylphosphate); (b) the ribonucleases of the liver and spleen, and of fractions B of the serum and urine, with a pH optimum of 7, are less sensitive to Zn2’ and Cu2+, and exhibit negligible activity versus uridine 3’-(e-naphthylphosphate). Determination of the serum level of pancreatic-type ribonuclease activity, with the use of uridine 3’-(cr-naphthylphosphate) or RNA as substrates, appears to be a valid diagnostic tool for pancreatic fibrosis in children. Introduction The alkaline ribonucleases of the rat which are relatively thermostable in nrid m_edi1um_ m_ay be divided in-to two @_mlJps: cmvotnrv P”qTfl_($ Qrigifi&q _“__ --------J in the pancreas and the salivary glands, and active vs RNA and the synthetic substrate uridine 3’-(a-naphthylphosphate), Up-naphthyl, and the enzymes from the remaining organs, such as the liver and spleen, which are inactive versus Up-naphthyl [l] . The pancreatic and salivary gland enzymes are secreted to the intestine, where they undergo partial absorption to the serum [2] , and

* Address correspondence to: Dr. Hslina Sierakowska. Institute of Biochemistry Academy of Sciences, 36 Rakowiecka St.. 02-532 Warszawa, Poland.

and Biophysics.

232

then transfer to the urine and the lysosomes of the epithelial cells of tlie kidney convoluted tubules [ 1,3,4] . ^ -._. The existence of two such classes of RNAases in severai mammalian species raises the question as to whether it also embraces the acid-thermostable RNAases in man and, if so, whether this differentiation in substrate specificity bears any relationship to the two known classes of human RNAases which differ in pH optimum, molecular weight, and affinity for cations [ 5-81 . A study of possible differences in properties, including substrate specificity, of human RNAases appeared of interest also in terms of its potential utility in clinical diagnosis. Some claims have been advanced for the diagnostic value of the RNAase levels of physiological fluids in various pathological states in man, e.g. chronic granulocytic leukemia [9], uremia [lo] and, possibly, hyperthyroid activity [ll] , We describe below the isolation and characterization of two types of acidthermostable RNAases, secretory and non-secretory, from various tissues and ..L:,I_. il_ ._1____:-1--:--l lX..:-1_ _X?.___.^. _.__I _Li_.__.^i_ LpIlysluluglcal 1ILlIUS Ul IllaIl) ~IIU a~~tmip~s LU ULIIIZ~ LII~ SpeCifiCiiji Of ihe SWEtory type of RNAase (mucoviscidosis).

in the serum as a diagnostic

aid for pancreatic

fibrosis

Materials and methods Substrates Yeast RNA, obtained from British Drug Houses (Poole, Dorset, England), was purified as described by Bardori and Pamula [ 121 to yield a product with a mean length of about 35 residues. E. coli sRNA, B grade, and highly polymerized RNA from yeast, A grade, were obtained from Calbiochem (Sacramento, Calif., U.S.A.). Rat liver precursor rRNA (>28S) was kindly contributed by Dr. Z. Lasota of the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw. Thymidine 3’- and 5’-(p-nitrophenylphosphates) were purchased from Raylo Chemicals Ltd. (Edmonton, Alta., Canada); and cytidylyl-3’-+ 5’-adenosine and adenylyl-3’+ 5’uridine from Sigma (St. Louis, MO., U.S.A.). Cytidine 2’,3’-cyclic phosphate was prepared as described by Szer and Shugar [13] ; and uridine 3’-(o-naphthylphosphate) according to Kole et al. [14]. Enzymes A was -Pancwstir _________i__R.NAaw ___. ___L- _..__ a - nroduct ~-__-‘__-I of sigmai

The diazonium salts Fast Red TR and Fast Garnet GBC were both products of G. Gurr (London, England). Acrylamide, N,N’-methylene-bis-acrylamide, N,N,N’,N’-tetramethyl-l,Z-diaminoethane and riboflavin were obtained from BDH; and Sephadex G-75 and SE-Sephadex C-25 from Pharmacia (Uppsala, Sweden). Blood serum employed for enzyme isolation was kindly supplied by the Warsaw Blood Donor Centre. Urine was collected from healthy adults. Serum used for estimations of RNAase activity was obtained from blood collected by capillary tube puncture and centrifuged. Duodenal contents were collected in tubes immersed in ice. Human pancreas, liver and spleen were obtained from q,,+r\.,0.;-0 x2-f 9,4 11 k cllL.r;l nCC_rUcxllm) J-“+L cab n+ Ckr. ~X7,lnlr: U-n,-.;+,.1 :, ~XT,u,....r auwpaK:J, bllt: “Y “13RI Il”qJ,lklJ 111 “V cumlw.

233

Electrophoresis on polyacrylamide gel at pH 4.5 was performed as described by Reisfeld et al. [15] and Gabriel [16] and protein revealed by staining with Amido Black. RNAase active against Up-naphthyl was localized by incubation of the gel in 3 ml of 2-fold dilutedDavis buffer 1171, pH 7.8, containing 2 pmol substrate and 3 mg Fast Red TR; incubation times were 3-24 h at room temperature, the medium being changed at 2-3 h intervals, and the pH maintained at 7.8 by addition of 0.1 N NaOH with a micropipette. alblYfia3t: hl Anl__ c&lJl”lby -nt;.r;+.l .rnr0..n A .,rno h-7 :mfix.hat:-\m ;m 1 ml nf mr\A; ve:13u3 Q&T IL*Yn ““a3 rl,+,,rm;,,rl U1;l,e;LlllllltlCI “y IIICIULJCL~I”,, 111I ,111“I ,IIC;u,_ urn containing 0.5 ml Davis buffer [17] at the desired pH, and 0.1 ml of a 1% aqueous solution of RNA, for 30 min at 37°C. Alternatively, the ionic strength of the medium was increased by the addition of NaCl to the desired concentration. The reaction was terminated by the addition of 1 ml 1 N HCl in 76% ethanol previously cooled to -15°C. The mixture was kept on the ice-bath for 15 min, and the resulting precipitate removed by centrifugation at 4°C. The supernatant was diluted 6-fold and the increase in absorption at 260 nm measured relative to a control sample without enzyme. RNAase activity is expressed in pmol acid-soluble products formed during 1 h incubation [18]. Unless otherwise specified, RNAase activity was determined against low molecular yeast RNA (BDH, purified according to ref. 12). RNAase activity versus cytidine 2’,3’-cyclic phosphate and dinucleoside monophosphates was determined by incubation for 0.5-8 h at 37°C in 50 ~1 0.1 M Tris/HCl buffer, pH 7.8 or 8.5 and 0.1 M NaCl, containing 0.5 pmol substrate. The course of reaction was followed by TLC on Eastman 6065 cellulose sheets developed with isopropanol/conc. NH40H/H20 (7 : 1 : 2, v/v/v), the extent of hydrolysis being estimated qualitatively under a dark UV lamp. RNAase activity versus uridine 3’-(a-naphthylphosphate) was assayed at pH 7.8 according to Zan-Kowalczewska et al. [ 11. Phosphodiesterases I and II were estimated with the aid of thymidine 3’- and 5’_(n_nit,rnnhPnvlnhnfnh~t.~~~ w ..A’~..y..v--J ~y~~~‘r*~~“v~, z

were determined according [20]. Phosphomonoesterase

d,pscrjhed

by

P_azze!]

[j$j].

Dr\Tb_ase I x_d II

to the procedure given by Sung and Laskowski activity was assayed according to Bessey and Love

[211. Protein

was estimated

by the method

of Lowry et al. [22] .

Results The following procedure was employed for the isolation of acid-thermostable alkaline RNAases from human pancreas, duodenal contents, liver, spleen, serum and urine. All operations were conducted at 5°C. Because of the differences in pH optima of the isoiated enzymes (see beiow), activities of pancreas and duodenal contents were determined at pH 8.5, for the spleen and liver at pH 7, and for serum and urine at both pH values: Step I. (1000 X g supernatant): 20% aqueous homogenates of pancreas, liver and spleen were centrifuged for 10 min at 1000 X g. Step II. (Sulfonosalicylic acid extract): The supernatants from Step I, or the physiological fluids (duodenal contents, serum, urine), were treated for 30 min with 0.1 M sulfonosalicylic acid, and then centrifuged for 10 min at 3000 X g. The supernatants were brought to pH 5 with 1 N NaOH, and then dialyzed for

10

20

30

20

FRACTION

50

60

70

NUMBER

Fig. 1. SE-Sephadex C-25 chromatography of human pancreatic ribonuclease: 110 ml of dialyzed SSA extract. with 0.05 M acetate buffer: _~~____. was .__ deonsited _._ ___.__ on - R* -. -1 R_ rm _ . . X 3;s cm co!xumn previous!y equ%brated PH 5.2, and eluted with a gradient formed between equal volumes of 0.05 M acetate buffer, PH 5.2, and 0.05 M acetate buffer, PH 4.4, containing 0.6 M KCI. with a collection of 5-ml fractions. . . . . . ., absorption at 280 nm; p, ribnnuclease activity versus RNA expressed asA260nmdue to 5 ~1 effluent after a 30-min incubation.

20 h against 0.05 M acetate buffer Step III. (SE-Sephadex C-25):

pH 5.2. 110-120 ml of dialyzate from Step II was deposited on an 18 cm X 3.5 cm column of SE-Sephadex C-25 previously equilibrated with 0.05 M acetate buffer pH 5.2, followed by elution with a KC1 gradient as shown in Fig. 1.

A

/

“9 /

- f-5

I :

B0.4.5- 8 2

k

-1.0 -

P J g

2 Gi k

0.3-

2 ros 0.15 1

. FRACTION

..f4.~

50 NUMBER

60

~

70

Fig. 2. SE-Sephadex C-25 chromatography of human serum ribonuclease, sulfonosalicylic acid extract after Step II, carried out as described in the legend to Fig. 1. . , . . . ., absorption at 280 nm; -, ribonuclease activity versus RNA assayed at pH 8.5, and -. - ., at PH 7.0 (A260-due to 10 itI effluent after 30-min incubation).

235

lb

2b

3b Lb Sb FRACTION NUMBER

6b

7b

Fig. 3. SE-Sephadex C-25 column chromatography of urine pancreatic ribonuclease, sulfonosalicylic acid extract after Step II, carried out as described in the legend to Fig. 1. . . . . ., absorption at 280 nm; -, ribonuclease activity assayed at PH 8.5. and -. - .. at PH 1.0 (Avrn - ” ” n.zI due to 2 ~1 effluent after a 30-min incubation).

RNAase activities of pancreas, duodenal contents, liver and spleen were all eluted at a KC1 concentration of 0.43 M KC1 (Fig. 1); whereas serum and urine each gave two peaks at about 0.3 M and 0.43 M KC1 (Figs. 2 and 3). Fractions 33-44 and 30-42 of the serum and urine peaks, respectively, eluted at 0.3 M KCl, and dialyzed versus 0.05 M acetate buffer pH 5.2, are referred to below as peaks A. Step IV. (Rechromatography on SE-Sephadex C-25): Serum and urine RNAase activities contained in peak B (fractions 57-65 in Figs. 2 and 3) were dialyzed for 20 h versus 0.05 M acetate buffer pH 5.2, and then rechromatographed as in Step III, with results shown in Figs. 4 and 5. The individual steps of the purification procedure are summarized in Table I.

i0

3b FRACTION

i0

$0

60

7b

NUMBER

Fig. 4. SE-Sephadex C-25 rechromatography of serum ribonuclease (peak B. fractions 57-85) from SESephadex C-25 chromatography described in Fig. 2. Elution was carried out as in Fig. 1. . . . . . .. absorption at 280 nm; -. - ., ribonuclease aCtiVitYassayed at PH 7.0 (A260m due to 40 ~1 effluent after a 30-min incubation).

FRACTION

NUMBER

Fig. 5. SE-Sephadex C-25 ~chromatography of urine ribonuelease (peak B, fractions 57451 from SESephadex C-25 chromatography described in Fig. 3. Elution was carried out as in Fig. 1. . . . ., absorption at 280 am; - - ‘, ribonuclease activity assayed at pH 7.0 (n260nm due to 30 /.d effluent after a 30-mia incubation).

TABLE I PURIFICATION SCHEME FOR HUMAN RIBONUCLEASES Step I, supernatant (1000 X X) of physioIogicaI fluids; Step II, sulf~lnos~i~ylic arid extract; Step III, SESephadex C-25; Step IV, rechromatography on SE-Sephadex C-25. The total activity is expressed in limo1 RNA rendered acid soluble in 1 h at 37’C. The specific activity is expressed in @mol RNA rendered acid soluble in 1 h at 37’C per mg protein. Purification step

Source of RNAase -_

Pancreas Total activity Specific activity Duodenal contents Total activity Specific activity Liver Total activity Specific activity Spleen TotaI activity Specific activity Serum, pH 7.0 Total activity Specific activity Serum, pH 8.5 Total activity Specific activity Urine, pH 7.0 Total activity Specific activity urine, pH 8.5 Total activity Specific activity

1 ---

-.

II -___---

III ________II_-._

24

45800 590

19500 12500

22700 72

12400 210

4140 a270

19600 8

16700 270

9500 2560

43600 17

39300 610

22100 5400 Peak B 800 350 Peak A 4340 1600 Peak B 2110 12300 Peak A 26650 186600

61100

6300 0.7

3200 13

18900 2

9250 41

9800 3250

9700 8300

33800 11200

33000 17300

IV

Peak B 275 640

Peak B 854 120000

.__.-___..__

237

Activities given are those measured after dialysis of the effluents and their concentration with the aid of Ficoll, hence they are appreciably lower than those of the original effluents. Protein contents of the effluents, determined by UV absorption at 280 nm and by the method of Lowry et al. [22], are artefactually elevated due to the presence of traces of sulfonosalicylic acid, and decreased after dialysis. For pancreatic RNAase, the increase in specific activity was about 500-fold, with a recovery of about 30% of the activity. For the duo“,A P,.” I:..,,. ,,A Afin”1 ,,r\n+nm+r.th,. nnrY*c-,TmA;ne P:m.unn I..,-.%._ 1 nn ” --A ww7 UC;1101 L”llLc11lm bk‘r: b”r~cxy”“U”‘~ IlgLutx.3 WCLt: I”” A d‘lLl ~“/O) QllLl I.“1 ‘l”tx a,,u spleen about 300 X and 50%. Serum and urine gave two active peaks (Figs. 2 and 3); peak A purified 800 X from serum and 15 X from urine, and peak I3 900 X from serum and 10 X from urine. None of the isolated enzymes exhibited detectable DNAase I and II, phosphodiesterase I and II, or phosphomonoesterase, activities. pfi-dependence of enzyme activities With RNA as substrate, the optimal pH for the RNAases of the pancreas, duodenal contents, and peak A of serum and urine, was 8.5 (Fig. 6). For the activities from liver, spleen, and peak B from urine, the pH optimum was 7.0. The activity of peak B from serum exhibited a rather broad optimum, pH 7.0-8.5, suggestive of non-homogeneity of the preparation. With Up-naphthyl as substrate, the optimal pH for the activities of pancreas, and peak A from serum, was 7.8. Ionic requirements All the RNAase preparations, with the exception of that from urine, required a salt concentration (NaCl) of 0.2-0.3 M for optimal activity. By con-

Fig. 6. pH-dependence of activities of purified human ribonucleases, assayed versus RNA in a-fold diluted liver and spleen; O------C, serum, pancreatic and duodenal contents, l -, Davis buffers. o-.----o, *. urine. peak B. urine, peak A; A----serum, peak B: n-3 peakA; .-,

238 TABLE EFFECT

I1 OF

Additives

CATIONS

ColK!.

ON

TIIE

ACTIVITY

OP

PIJRIFIED

IIUMAN

RIBONUCLEASES


(mM) Pan-

Duodenal

cress

contents

Liver

Spleen

SelUItl Peak

Urine A

Peak

B

Peak

A

Peak

M&O4

l-5

0

0

0

0

0

0

0

0

CaC12

l-5

0

0

0

0

0

0

0

0

ZnS04

1

38

41

0

0

38

0

37

0

cuc12

1

16

18

0

0

17

0

17

0

EDTA

1-5

0

0

0

0

0

0

0

0

B

trast, the urine enzyme was virtually insensitive to salt concentration over the range 0.1-0.3 M. The influence of Zn*+ and Cu*’ on the activities of the various preparations is TT -....1:_11_. Z.-l-:L.:c CL --+:..:C:-.- P",, ____..,.,xn ShoWn in m-1_,_ rauie 11. Both CaiiOiiS pa-uauy IIIIIIU~L LII~ il~~~v~wz'b LLU~L pai~ucas, duodenal contents, and peak A of serum and urine, but are without effect on the remaining enzymes. None of the enzymes were affected by Mg” and CaL’, or by EDTA at concentrations up to 5 X 10e3 M. Enzyme thermostability Heating the various enzyme 15-45s decrease in activity. these conditions.

preparations for 5 min at 100°C at pH 3 led to a Only the spleen enzyme was unaffected under

Activities versus RNA, dinucleoside monophosphates and Up-naphthyl The specificity of human ribonucleases generally resembles that of the bovine pancreatic enzyme. Human RNAases exhibit activity versus cytidine 2’,3’-cyclic phosphate and, at a much higher rate, versus cytidylyl-3’+5’adenosine. Adenylyl-3’+5’-uridine is resistant; even a lOO-fold excess of enzyme over the amount necessary for hydrolysis of cytidylyl-3’+5’-adenosine does not cause detectable hydrolysis of adenylyl-3’+5’-uridine. During the course of this study, it was noted that human RNAases hydrolyze different RNA preparations at different rates. For example, with human pancreatic RNAase, the relative rates of hydrolysis are: (assuming that the rate for purified BDH yeast RNA is l), 0.16 for rat-liver pre-rRNA (>28S), 0.56 for Calhinrhnm UI”YllL.lll

hiuhlv “‘b”LJ

nnlwnori7od y--J III._,LIYIU weact J VU”.. RNA &..A.AA) 0.8

for C’nlhinrhem F co!i &N-A_, .J.+--* ______--_i.

No

attempts were made to establish whether these differences in rates were due to differences in RNA structure (such as differences in extent of double-strandedness) or to differences in cation contaminations. For this reason, and as mentioned in Materials and methods, most measurements in this study involved the use of only one RNA preparation. The human RNAase samples exhibited different rates of hydrolysis of Upnaphthyl (Table III), e.g. the ratio of the rates of hydrolysis of RNA and Upnaphthyl was 17-28 for the enzyme from pancreas, duodenal contents and peak A of serum and urine. For the enzyme from liver, spleen and peak B of urine, this ratio was well above 400. For peak B of the serum, which is contam:-nt,rl ..r;Ch n.~m,m-no+;,, ,,nnr,mn +hn rq+;,, X,,OP 919 IllabtTu ""Ib,II pallblr;auL r;ll~yIllr;, b,Ic; La"," ""CIU YI".

239 TABLE

III

ACTIVITIES AND

OF

PURIFIED

HUMAN

RIBONUCLEASES

AND

BOVINE

RNAase

A VERSUS

RNA

Up-NAPHTHYL

Ribonuclease

preparation

Activity

versus

RNA (in km01 acid-solubilized substrateihlmg protein) Human pancreas Human duodenal contents Human liver Human spleen Human serum Peak A Peak B Human urine Peak A Peak B Bovine pancreas -RNAase A

12500 8270 2560 5400

Up-naphthyl (inn01 hydrolyredlhlmg protein)

Ratio: activity versus RNA/activity versus Up-naphthyl

..-450 460 3 5

28 18 854 1080

640

85 3

19 213

166600 >20000 590000

9800 50 5000

17 >400 118

1600

Gel electrophoresis of isolated RNAases The electrophoretic mobilities on polyacrylamide gels of the isolated enzymes, as revealed by the amido black reaction for protein and activity versus Up-naphthyl, are exhibited on Plate 1. It will be seen that the enzymes from

a

b‘

c

d

Plate 1. Electrophoresis on polyacrylamide gel of human purified (a) pancreatic, fb) duodenal contents, te) serum and fd) urine rlbonucleases. I, protein stained with Amido Black; II, activity revealed versus Up-naphthyl as described in Materials and methods. Cathode at the bottom.

TABLI,:

I\’

KIBONUCLEASE

LEVELS

IN

Number

Organ

ADULT

HUMAN

ORGANS

Activity

of

(m ~.lmol RNA

individuals

solubili~ed/h/mm

tested

(hIem

I

acid

tissur)

S.D.)

pancreas and duodenal contents are electrophoretically homogeneous and active against the synthetic substrate. Peak A of serum and urine exhibited two protein-positive bands with almost identical mobilities, and active versus Upnaphthyl; but it did not prove possible to establish whether the activity resided in one or both of the bands, because of the poor resolution. RNAase activities in adult organs Table IV shows that the RNAase activity of the adult pancreas is only about 1.2 pmol acid-solubilized RNA/h/mg tissue, i.e. about 20% higher than that of liver, and approximately 50% that of the spleen. RNAase levels of serum and duodenal contents in normal children and in cases of pancreatic fibrosis The existence in human serum of pancreatic-duodenal type RNAase activity, and the ability to specifically determine the level of such activity with the aid of Up-naphthyl as substrate, prompted us to examine the activity of this enzyme in children suffering from pancreatic fibrosis. Table V exhibits the RNAase activities, measured against RNA and Up-naphthyl, of normal children 2nd nf PRCPQ I._. invnlvinu nnnrwmtir fihrncic l?nr nnrmal rhildmn RNAaw _I_ VI _U”V” V..“.b y-.“I.,uY’~ LIU&VUI”. L VL the Y.&YLlVllllUl Y”“U’Y”) AYL. I lU”V activity decreased with growth, the decrease being more marked for the pancreatic-type enzyme than for the total RNAase activity, measured versus RNA. By contrast, children afflicted with pancreatic fibrosis exhibited a considerably lower level of activity relative to a control group of the same age.

TABLE

V

RIBONUCLEASE PH

7.8)

IN

THE

ACTIVITY, SERUM

OF

DETERMINED NORMAL

Number

A@

AGAINST

CHILDREN

cases

of

RNA

AND Activity

OF

(AT CASES

versus

RNA

(bum01 acidsolubiked

pH

8.5)

AND

WITH

Number cases

UP-NAPHTHYL

PANCREATIC of

Activity

substrate/h/ml)

(firno h/ml) (Mean

Normal

hydrolyzed/ i S.D.)

children 52

196

t 38

23

6.8

+ 2.4

47

157

+ 41

19

3.2

+ 0.5

years

39

131

’

27

21

2.9

’ O.fi

years

12

60

k 17

12

1.1

IO.4

1-7

days

l-12

months

2-7 Cases

versus

Up-naphthyl

(Mean

+ S.D.)

of pancreatic

fibrosis 24

(AT

FIBROSIS

241

TABLE

VI

RIBONUCLEASE PH

7.8)

CREATIC

IN

THE

ACTIVITY,

DETERMINED

DUODENAL

CONTENTS

AGAINST

RNA

OF

NORMAL

of

Activity

(AT

pH

CHILDREN

Age

Number

(Years)

cases

Up-NAPHTHYL

OF

CASES

WITH

(AT PAN-

versus Up-naphthvl

RNA

h/ml)

solubilized/

(/an01

hydrolyzed/

h/ml)

children 2-7

Cases

AND

AND

FIBROSIS

(&&no1 acid

Normal

8.5)

of pancreatic

18

482

12

91

+ 116

7.8

+ 2.8

i

1.3

? 0.5

fibrosis 2-6

53

The decrease in activity versus RNA almost parallels that versus Up-naphthyl, which is consistent with the fact that pancreatic-type RNAase constitutes about 80 percent of total serum activity and the non-pancreatic component exhibits only 40 percent of its activity at pH 8.5. An even more pronounced decrease in RNAase level occurs in the duodenal contents of afflicted children (Table VI) ; the level of activity (only the pancreatic-type enzyme is present in the duodenal contents) decreases more than 5-fold against both substrates. Discussion The foregoing results demonstrate clearly that the acid-thermostable alkaline RNAases in man may be divided into two classes which differ in their optimal pH, behaviour towards Zn” and Cu2+, and activity towards the synthetic substrate Up-naphthyl. One class exhibits a pH optim_um_ of 8.5, inhibition hv Zn2+ 2nd Cuz*, and -.? --activity against Up-naphthyl. It includes the RNAases of pancreas, duodenal contents, and a portion of the activities of serum and urine (peaks A in Figs. 2 and 3). The second class of enzymes is optimally active at pH 7, is not affected by 10e3 M Zn2+ and Cu’+, exhibits minimal or no activity versus Up-naphthyl, and is found in the liver and spleen and, also, along with the first class, in serum and urine (peaks B in Figs. 2 and 3). These two types of RNAase undoubtedly correspond to those isolated from human urine by Delaney [ 51. One of these, with a pH optimum of 8, possesses the physico-chemical and immunological properties, as well as the specificity, of bovine pancreatic RNAase. The other, with a pH optimum of 6.5, cioseiy resembles human spleen RNAase. Two types of thermostable RNAase, with different pH optima, and sensitivity towards Zn2+ and Cu’+, were also found in human serum and urine by Naskalski [6,7] and Sznajd [8]. Differences in secretory and non-secretory properties of acid-stable alkaline RNAases have also been reported amongst other mammalian species [1,18,23, 241 . These differences include immunological properties, pH optima, molecular weights, affinities towards cationic resins, sensitivities toward8 Zn2+ and Cu’+, and relative activities towards Up-naphthyl.

The occurrence in human serum and urine of RNAases with properties resembling those of pancreatic RNAase, as well as those of liver and spleen, suggests t,hat their origin is not only the pancreas and salivary glands, but also other organs. The presence of pancreatic-type enzyme in the serum and urine of mammals can be explained by its transport from the duodenum into the blood stream [2] and then to the urine [25] , By contrast, little is known about the transport to these fluids of other acid-stable RNAases, such as those of liver Il?rf Yy’.,c”, clllclnn UA”‘.“Uh” althntf “L thn v.(i._ UIl“&.LUl ty in properties of both groups of enzymes. The ability to specifically evaluate the level of pancreatic-type RNAase in serum with the aid of Up-naphthyl as substrate points logically to its possible use as a diagnostic tool in pancreatic insufficiency or duodenal malabsorption. Initial attempts to check this involved cases of pancreatic fibrosis in children, manifesti~~g itself in pancreatic exocrine insufficiency. Diagnostic tests hitherto applied to the detection of pancreatic fibrosis are based on the determination of electrolyte levels in perspiration, and activities of trypsin, amylase and lipase in the duodenal contents [ 261 . The results of the present investigation clearly demonstrate that, in established cases of pancreatic fibrosis, then: is a more than &fold decrease in RNAase activity of the duodenal contents (with either RNA or Up-naphthyl as substrate), and a somewhat, smaller decrease of pancreatic-type RNAase activity in the serum. Since assay of serum activity versus Up-naphthyl reflects changes only in pancreatic-type RNAase activity, it is a potentially more reliable indicator of exocrine pancreatic function than assay versus RNA, which includes fluctuations in the non-pancreatic-type RNAases. Such fluctuations in nonpancreatic-type RNAase levels have been reported in the urine in cases of chronic granulocytic leukemia by Naskalski [ 91. The sensitivity of Up-naphthyl as a substrate is such that only 10 ,IL~serum, collected. by rilnillnrv tube nllnctilre is rm~lirfd This ~enrwwnt,s 3 cQnsid.erab!e r-____-__, -I __l-____‘. --L---‘--J r^------improvement over the present procedure, which requires the collection of the duodenal contents for determination of the activities of pancreatic enzymes, and which is particularly difficult in cases of childhood pancreatic fibrosis where the quantity of duodenal contents is not only very low, but the material itself is quite viscous. Decreases in the level of pancreatic type RNAase activity in serum may be expected also in other diseases, such as pancreatopathy, malabsorption, etc. The diagnostic test for the level of serum pancreatic type RNAase activity is consequently not specific for pancreatic fibrosis. It is, nonetheless, a simple test complementary to observed clinical symptoms and to determinations of elec1-..-,_.L-_ :._ II._ -_..--:.__L:-... Lrulylxb III Llle perspu-auur1. It should be noted that serum RNAase levels have been examined under a variety of pathological states. However, with the exception of granulocytic leukemia, no attempts were made to distinguish between pancreatic and other types of RNAase activity. Pronounced deviations from the norm, usually an increase in activity, have been noted in cases of disseminated hepatic carcinoma [ 271, uremia [lo], chronic granulocytic leukemia [9] and hyperthyroidism

1111. The level of serum RNAase activity

in normal healthy

adults falls within the

243

range 0.08-0.44 pg pancreatic-type enzyme per ml [25,28-311. This corresponds to the value found in children in the age-group 2-7 years (130 pmol RNA rendered acid soluble/h/ml serum corresponds to 0.21 pg bovine pancreatic RNAase/h/ml serum). The variations in the values presented by different authors for the levels of human serum RNAase activities are undoubtedly due in part to the use of different RNA substrates (see above) and different ionic strengths of the incubation medium, the human RNAase being highly sensitive to both these factors. Finally, particular attention should be drawn to the remarkably low level of RNAase activity in the human pancreas, lower even than that in the spleen (Table IV). This is in agreement with the suggestion of Barnard et al. [32] that human pancreatic RNAase does not fulfil an essential digestive function. Acknowledgments 1x7,.

vvt:

utqJ1y rl-_-I__

^Y^

ilIe

:-rl_LL_-l

I_

I”u’o_wako_wska

A*

TX..

w

111ueIJleu

j-jr*

aiid

R.

for

t’v’ino.wska

UT.

making the human tissues and body fluids available; and to Mr. J. Sosnowski for excellent technical assistance. This investigation was carried out with the partial support of the Polish Academy of Sciences (Project 09.3.1). References 1

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