371

Clinica Chimica Acta, 65 (1975) 371-377 @IElsevier Scientific Publishing Company,

Amsterdam

-Printed

in The Netherlands

CCA 7475

PROPERTIES OF fl-GLUCURONIDASE HUMAN SYNOVIAL FLUID

L. MORO,

B. DE BERNARD

ACTIVITY

IN

* and F. GONANO

Istituto Regionale Medicina Fisica e Riabilitazione, Laboratorio di Patologia Clinica, Udine (Italy) (Received

July 26,1975)

Summary The purpose of the present study was to evaluate the properties of pglucuronidase (EC 3.2.1.31) in human synovial fluid. It was shown to have a pH requirement of 5.0 and a KM value of about 8.0 * 10e3 M using phenolphthalein /3-glucuronide as the substrate. At low substrate concentration an endogenous inhibitor is demonstrable. The inhibition is of the competitive type and is removed by proteolytic digestion of synovial fluid, whereas hyaluronidase digestion and addition either of Triton X-100 or of various salts to the assay mixture, are ineffective. The possibility that the inhibitor is a protein from serum is discussed.

Introduction The role of lysosomes in tissue injury, in general, and in connective tissue diseases, in particular, has awakened a great interest in the hydrolases of synovial fluid [l-4]. These enzymes may play an important role in the biochemical mechanism involved in joint disorders. fl-Glucuronidase is directly related to glycosaminoglycan catabolism, since it removes terminal non-reducing residues of glucuronic acid from the oligosaccharides produced by hyaluronidase activity on hyaluronate or chondroitin sulfates [5]. A good correlation between release of /3-glucuronidase and rate of bone resorption is reported by Vaes [6]. It appears therefore important to study the properties of this enzyme in synovial fluid, in an attempt also to offer to clinicians an enzyme assay useful to evaluate the type of joint disease, the efficiency of therapeutic treatments and which is easier to carry out than that for hyaluronidase activity. During the classical

* Permanent address: Istituto di Chimica Biologica. University degli Studi di Trieste. Italy.

312

approach to determine the kinetic natural inhibitor was found. Experimental

properties

of the enzyme,

the presence

of a

Procedures

Enzyme sources Synovial fluids were aspirated under aseptic conditions from the knee joint of patients with rheumatoid arthritis or osteoarthrosis and either immediately used or frozen at -20”. Before assay, the synovial samples were thawed and freed of cellular elements by centrifugation. fl-Glucuronidase from snail juice was obtained from Endo (U.S.A.) (glusulase); bacterial protease (Streptomyces griseus) from Sigma (U.S.A.) and hyaluronidase from Miles-Seravac (U.S.A.). The post-mitochondrial fraction was used as the source of /3-glucuronidase from rat liver. Assay conditions The procedure was derived from the method of Talalay et al. [7] modified by Kerr and Lewy [8]. The optimal microassay system had a volume of 200 ~1 containing: 25-100 ~1 of synovial fluid; 5mM phenolphthalein glucuronide (Sigma, U.S.A.) in 0.1 M acetate buffer, pH 5.0; 1 ~1 of a solution containing 40 mg/ml of gentamycin sulfate (Schering Corporation, U.S.A.) and 0.1 M acetate buffer pH 5.0, to complete the assay volume. The incubation time was 60 min at 37”. The reaction was stopped by adding 600 ~1 of 0.133 M glycine/NaOH buffer, pH 10.45. After centrifugation at 6000 X g for 2 min, the amount of phenolphthalein released was measured at 543 nm in a LKB 2074 calorimeter. The activity is expressed as pmol of phenolphthalein released per ml of synovial fluid per h. Assay of inhibitory activity was carried out as follows: various amounts of synovial fluid were preincubated for 3 min with fl-glucuronidase of the glusulase preparation or of the liver post-mitochondrial fraction. The residual activity of the enzymes was then measured as described above. 1 unit of glucuronidase activity is defined as that amount of enzyme which will produce 1 pg of phenolphthalein in 1 h at 37”. Analytical procedures Digestion of synovial fluid with hyaluronidase or with bacterial protease was performed as follows: 0.5 ml of synovial fluid was incubated at room temperature for 20 h, in a small dialysis bag, either with 0.1 ml of 0.1 M acetate buffer, pH 4.5, containing 0.15 M NaCl and 10 mg/ml hyahrronidase or 0.1 ml of 0.1 M phosphate buffer, (pH 7.5), containing 10 mg/ml protease. Dialysis was performed first against the buffer solutions and then exhaustively against deionized water. The activity of the bag content was then assayed as indicated above. Protein was determined by the Biuret method 191. Results In a series of preliminary experiments, sured in synovial fluid either in the presence

p-glucuronidase activity was meaof 0.1% Triton X-100 or following

313

5

6

i

pH

Fig. 1. Effect of PH on P-glucuronidase activity of human synoti fluid. The buffers used were 0.1 M acetate, (PH 4.0-5.6, X X): 0.1 M citrate, (PH 4-6.2. l __ a); 0.1 M phosphate buffer, (PH 5.8-7.6, o 0). Enough NaCl was added to correct the ionic strength. c

s:

$ ‘2 0.16

?s ,6 ‘5 ?e

I

+Iv

18

ZP r’7 gt

0.06

12

sy ‘la E 3

Km =0.62

J-.

10m3M

//. 2.5 phenolphtaleln

5.0

Fig. 2. The relationship of product l/V versus l/S. Incubation conditions

Fig. 3. The relationship of product mM phenolphthalein fl-ghxuronide. of the enzyme assay mixture.

3.2

1.6

p -Glucuronide

CmM)

formation to substrate concentration, as described under methods.

l/S

(mM)-’

with an inserted

formation to svnovial protein concentrations, See under methods the other assay conditions.

K~

plot

of

in the presence of 0.25 Activity refered per ml

374 TABLE

I

SNAIL

JUICE /I-GLUCURONIDASE

MAN

SYNOVIAL

ACTIVITY

IN THE ABSENCE

AND

IN THE PRESENCE

OF HU-

FLUID

Enzyme solution = 3000 units/ml. Values are expressed as nmol of phenolphthalein released/ml of the assay mixture/h. Mean of 4 experiments fS.D. Synovial fluid (fill

-

Activity

% inhibition

P

-

157.5 t. 25