Bovine thyroidal xanthine oxidase

BOVINE

THYROIDAL HYO-SA

Biochemistry

Department.

North

LEE*

Dakota

XANTHINE ALLAN

and State

(Rewired

G. FISCHER~

University.

9 Mrud~

OXIDASE’

Fargo.

North

Dakota

58102. U.S.A

1978)

Abstract-l.

Xanthine oxidase from bovine thyroid glands was purified IOOO-fold. Electrophoresis of the purified enzyme yielded a single enzymatically actice band. 2. A molecular weight of 260.000 + 20.000 was estimated by gel filtration and by polyacrylamide gel electrophoresis. 3. By electrofocusing the isoelectric point was shown to be 6.2 and the enzyme has a pH optimum at 7.8. 4. Among the most common substrates, thyroid xanthine oxidase oxidizes only xanthine and hypoxanthine with oxygen as an electron acceptor. 5. With dichloroindophenol as the electron acceptor the preferred substrates are xanthine and hypoxanthine compared to NADH. NADPH and acetaldehyde. 6. Based on the isolation procedures and the K, and V,,,,,, values. thyroidal xanthine oxidase appears to be present in sufficient concentration to supply the hydrogen peroxide requirement of the thyroid gland.

INTRODUCTION

In a previous publication (Fischer & Lee, 1973) we reported the presence of xanthine oxidase in bovine thyroid glands and have since obtained data to indicate a possible physiological function for the enzyme (Lee et nl.. 1977). Xanthine oxidase catalyzes the oxidation of either hypoxanthine or xanthine to form uric acid and hydrogen peroxide. It is the hydrogen peroxide which may act as the oxidant for iodide (DeGroot & Cavalli, 1960) and the coupling of iodotyrosine molecules to form the iodinated thyronines (Fischer ef ul., 1964). Recently we published data showing the thyroid gland to contain a purine nucleoside phosphorylase which supplies the substrates for xanthine oxidase (Moyer & Fischer, 1976). This report describes the purification of thyroidal xanthine oxidase and indicates some of its properties as related to xanthine oxidase isolated from other tissues. The enzyme has a wide tissue distribution. Isolation and purification procedures have been given for liver (Kielly, 1955). intestine (Roussos, 1963), duodenal (Duke et al., 1973), milk (Massey er al., 1969), brain (Markley er al., 1973), and more recently from mouse skeletal muscle (LaLanne & Willemot, 1975). This work also suggests that the thyroidal xanthine oxidase is present in sufficient concentration to supply the hydrogen peroxide requirements of the gland. EXPERIMENTAL

PROCEDURES

Materials

Bovine thyroid glands were a gift of Flavorland Industries, Inc., West Fargo, North Dakota. Acrylamide, bisacrylamide and N,N,N’,N’tetramethylethylenediamine were obtained from Eastman. Sephadex G-25 and G-200

’ Journal Article Number 864. North Dakota tural Experiment Station. ’ Present address: Department of Biochemistry, University Medical Center. Indianapolis, Indiana. 3 To whom reprint requests should be sent.

AgriculIndiana

559

were supplied by Pharmacia. 2.6-Dichloroindophenol was obtained from Matheson. Coleman and Bell: apoferritin and N-ethylmaleimide from Mann Research Laboratories. All other biological chemicals were purchased from Sigma Chemical Co.

Xanthine oxidase assays in which oxygen was the electron acceptor have been described previously (Fischer & Lee. 1973). However, when artificial acceptors were used. the activity of the xanthine oxidase was measured as follows in the presence of 66 {IM xanthine and 50 mM phosphate. pH 7.8: dichloroindophenol (40pM) reduction. by the decrease in absorbance at 600nm: cytochrome ( (2OpM) reduction, by the increase in absorbance at 550nm: nitro blue tetrazolium (40pM) reduction, by the increase in absorbance at 535 nm; phenazine methosulfate (50pM) reduction. by the aerobic formation of uric acid as determined at 292 nm (Kanada et al., 1972). Protein concentrations were determined by either of two methods. For the higher concentrations. the protein was measured by the biuret method (Gornall et al.. 1949) and lower protein concentration determinations were made by the method of Lowry er al. (1951). All spectrophotometric determinations were performed on a Perkin-Elmer Coleman Model I24 Recording Spectrophotometer. Molecular

weight

determinations

The molecular weight determination of the enzyme on Sephadex G-200 was made according to the method of Andrews (1964) and Whitaker (1963). All operations were carried out at 4-6’C. One-half mg sample of each marker in 2.0ml of 0.1 M phosphate. pH 7.4 was applied sequentially in descending order of molecular weight to a column (1 x 78 cm). The samples were eluted at a constant flow rate of I.5 ml/20 min/tube. The absorbancy of the effluents was measured at 280 nm. Molecular weight markers used were: apoferritin (480,000), a-globulin-human (160,000), serum albumin (67,000), egg albumin (45,000). Blue dextran 2000 (2,000,OOO) was used to determine the void volume of the column. Molecular weight of the xanthine oxidase was also estimated electrophoretically by the procedure of Hedrick and Smith in a Buchler polyanalyst electrophoresis apparatus

560

HYO-SA LEE and ALLAN G. FISCHER

(Hedrick & Smith. 1968). The gel system consisted of 0.4 ml of the concentrating gel on top of 0.6 ml of the resolving gel in 6 x 75mm tubes. The concentrating gel with 2.5:” acrylamide monomer was photopolymerized and the pore gel concentrations of the chemically polymerized resolving gel varied between 6 and 117;by the dilution of a concentrated solution of 3O”b acrylamide monomer. The ratio of bisacrylamide to acrylamide monomer concentration was kept constant for the variation of the resolving gel concentrations. The following standard proteins were applied as molecular weight markers: thyroglobulin (680.000), milk xanthine oxidase (290.000), y-globulin (156,000).bovine serum albumin 168,OGO)and egg albumin (45,000). The 50~1 samples containing 50 fig of protein were electrophoriz~ at room temperature at 2.5 ma per tube. The samples contained bromophenol blue as a tracking dye and sucrose to increase their density. At the termination of the run, the gels were removed from the tubes and stained with O.l”;, aniline blue black and destained electrophoretically at 4ma per tube with 7%, acetic acid. Migration of the dye and proteins was measured and the data presented as in the original article (Hedrick & Smith. 1968).

This was accomplished by the procedure of LKB in an LKB 8101 Ampholine electrofocusing apparatus. The column of the electrofocusing equipment was sequentially filled with the following solutions: I5 ml of anode solution (0.2ml 85th HsPO,, 12g sucrose in 15.0ml HzO). llOm1 of the ampholine (pH 5--8) sucrose solution prepared with 55 ml of a light solution (1.6?,, ampholinej. 55 ml of a dense solution (5.29,, ampholine in 700; sucrose) and IOml of a cathode solution (0.8 g NaOH in.10 ml HZO). The 55 ml of the light solution contained 1Omg of the enzyme which had been dialyzed against 0.005 M Tris-HCI, pH 8.1. The electrofocusing experiment was carried out at O’C and the maximum power did not exceed 3 W throughout the run of 48 hr. The column was emptied from the bottom at a flow rate of 1.7ml/min/tube. The effluent was checked spectrophotometrically at 280nm for protein and at 292 nm for enzyme activity and the pH measured with an Orlon Model 701 digital pH meter.

60 mM Tris-glycine,

pH 8.3 at 490 V and 4 ma in the cold room. The apparatus was constructed from the original design of Whitehead et al. (1971). After a ueriod of 14 hr. the current was disconnected and the l&t tan band of xanthine oxidase was eluted with the same buffer. The fractions containing xanthine oxidase were combined and concentrated by ultrafiltration using a PM-10 membrane. Prepurarice

Preparazice

colunm

electropharesis

on

Sephade.x

G-2.i.

The above concentrated enzyme was applied to a Sephadex G-25 column (2.2 x 40cm) which was equilibrated and electrophorized under a continuous buffer system of

yei electraphoresis.

This pro-

Poly-Prep apparatus using the electrophoresis system developed by Ornstein & Davis (1962). A photopolymerized 2.5”,, gel (40 ml) was cast over a chemically polymerized 7.5”, gel (60ml). The electrophoresis buffer system consisted of 0.1 M Tris-HCI. pH 8.1 as a lower buffer and 0.053 M Tris-glycine. pH 8.9. as an upper buffer. After an electrophoresis period of 12 hr at 40 ma and O’C the resolving gel containing the enzyme was extruded from the column and a thin vertical strip was cut from the gel. The location of xanthine oxldasc was determined by treating the strip of gel with 0.1 mM nitro blue tetrazolium dye and 5OpM xanthine in 5OmM phosphate, pH 7.8. The remaining portion of the gel coniaining the active xanthine oxida& i*as macerated & 0.1 M Tris-HCI. pH 8.1. The eluted enzyme was separated from the gel by filtration through fine glass wool.

RESULTS

A summary of purification statistics for a typical puri~cation of thyroidal xanthine oxidase is given in Table 1. The procedures were consistent in separating an enzyme with a specific activity of about 1,000 from several isolations. Eiectrophoresis

When the enzyme which was eluted from the preparative gel electrophoresis step was eiectrophorized and the gels developed with amido black or nitro blue tetrazolium. only 1 band was detected in each gel (Fig. 1). Isoelecfric

Fresh bovine thyroid glands were homogenized and the thyroidal xanthine oxidase was partially purified as described (Fischer & Lee, 1973). The previous steps included pancreatic digestion, ammonium sulfate precipitation and bulk calcium phosphate gel adsorption. The enzyme that was eluted from the calcium phosphate gel was concentrated by an Amicon Ultrafiltration apparatus (Model 52) with an XM-1OOA membrane under 25 psi of N2 pressure. The concentrated enzyme was dialyzed overnight against 0.t M phosphate buffer, pH 6.2, containing 1mM salicylate and 0.3 mM EDTA. Calcium phosphate-cellulosr chromatogruphy. A mixture of calcium phosphate and cellulose was prepared by mixing 500 ml of calcium phosphate gel (dry weight. 20 mg/mlj and 67g of cellulose (Whatman CF II) in 500ml of H,O. The dialyzed enzyme obtained from 2 I of thyroid supernatant was applied to a calcium phosphate-cellulo~ column (I x 30 cm) which had been equilibrated with the same buffer as the enzyme had been dialyzed against. The column was eluted with the dialyzing buffer until all the colored impurities had been removed. The enzyme was then eluted by the same buffer containing 5% (NH&JO,. Fractions (3-m]) containing enzyme activity were combined and concentrated again by ultra~ltration using a PM-IO membrane.

polyacrylamide

cedure was carried out in a Buchler

point

Analysis for oxidase activity and absorbance at 280 nm in the fractions after elution from the ampholine column in a pH range of 5-8 revealed only one protein peak which coincided with the one xanthine oxidase peak with a pI of 6.2 (Fig. 2). Molecular

weight

different techniques were used to estimate the molecular weight of the thyroid enzyme. gel filtration and analytical gel electrophoresis. Five proteins were used to calibrate the Sephadex G-200 column including milk xanthine oxidase. The elution pattern of purified thyroidal xanthine oxidase on Sephadex G-200 suggests a molecular weight of about 240,000. Using analytical gel electrophoresis with 6 different proteins, including milk xanthine oxidase and calculating the data according to Hedrick & Smith (1968) a molecular weight of approximately 280,000 is suggested for thyroidal xanthine oxidase. Two

Substrate

sprcjficitj

The most common substrates and electron acceptors for xanthine oxidase were studied to determine the substrate specificity of thyroidal xanthine oxidase. The rates at which several electron acceptors are reduced by the substrates (xanthine, hypoxanthine, NADH and NADPH and acetaldehyde) were deter-

561

Fig. I. Polyacrylamide wth 0.1 mM nltro blue partially puriticd cnzymc phoresis of the ewyme aniline blue black dye. with nitro

gel electrophoresis of thyroid xanthine ouidase. o-Thin vertical strip stained tetrarolium dye and 5OitM xanthine in 50mM phosphate (pH 7.8) after the was clectrophorized on the preparatlvc gel for 12 hr. I-Analytical gel electroeluted from the preparative gel electrophoresls step. stained for protein wth I’-Analytical gel electrophoresis of the same enzyme preparation but treated blue tctrazolium dye and xanthine for wanthine oxidase activity.

Bovine thyroidal Table

I. Summary

Fraction Supernatant Pancreatin digest 20”. n-BuOH 35-55”” (NH&SO4 Dialysis Cal-P-gel UF XM-100 A Cal-P-cellulose G-200 column Prep G-25 electrophoresis Prep PAG electrophoresis

of results

xanthine

for the purification

Total volume (ml)

Total protein (mg)

4.000

IY8.000

Total units.’

563

oxidase of thyroid Specific activityh ( x 10-4)

xanthine

Recovery (“J

Fold puriiication

loo.0

I

I

19.8

oxidase

395 130 11 I6 15

5.530 342 274 143 83

10.5

6.4 6.5 4.7 4.9

I9 187 237 330 590

53.0 32.3 32.8 23.7 24.8

19 187 237 330 590

I5

64

4.8

743

24.2

743

24

2.5

1.030

17.6

I.030

4.5

“A unit of enzymic activity was defined as the number of itmoles of uric formed per min at 25’C with 66.6jcM xanthine as substrate. h Spectfic activity of the enzyme was expressed as units per mg of protein.

mined over several substrate concentrations. The apparent K, values obtained from a double reciprocal plot of the data according to the method of Lineweaver & Burk (1934) are given in Table 2.

Enzyme activity of thyroid xanthine oxidase was studied over a pH range of 6.2-9.0 using 0.1 M phosphate for the lower range and 0.1 M Tris for the higher range. The enzyme was maximally active at pH 7.8 with xanthine or hypoxanthine as the substrate, but shows a broad peak of activity between 7.4 and 8.2 with only approximately 10% difference in maximum activity between those values and pH 7.8. E&W

qf’curious

additives

on enzyme

actioir)

The results of various additives on the enzyme activity are summarized in Table 3. The sulfhydryl reagents. p-hydroxymercuribenzoate and N-ethylmaleimide, strongly inhibited the enzyme activity while sodium arsenite had no inhibitory effect at levels tested. Antithyroid drugs, tapazole and thiourea, exhibited little inhibition of the enzyme. Parnate (2-phenylcyclopropylamine). a potent inhibitor of thyroidal monoamine oxidase (Fischer et a/., 1967). exhibited no inhibition. The thyroid enzyme was not readily inhibited by the metal complexing reagents, EDTA, sodium azide, and sodium cyanide. However, diethyldithiocarbamate, neocuproine, cupferron, and 2:2’-bipyridine exhibited strong inhibition effects. DISCL’SSION

Xanthine oxidase has been isolated from bovine thyroid tissue and appears to be electrophoretically pure. The initial steps of a procedure of Massey et al. (1969) which were successful in isolating xanthine oxidase from milk appeared to be satisfactory through the calcium phosphate-cellulose column step. A digestive step with pancreatin was necessary to obtain an active enzyme from the thyroid non-partiR.C.9t-B

acid

culate fraction that could be precipitated with ammonium sulfate. Further purification of the enzyme by DEAE-cellulose chromatography according to a method developed by Roussos (1967) or variations thereof was fruitless, as poor recovery of the enzyme always resulted. As an alternative procedure, preparative column electrophoresis on Sephadex G-25 was introduced for further purification of the enzyme. This step resulted in only a 1.2-fold purification but some dark material was removed which tended to interfere with the next step, preparative polyacrylamide electrophoresis. Elution of the enzyme from the gel with a reasonable recovery of the activity and reproducibility of the procedure was unsuccessful by the standard elution procedure which requires complete electrophoresis of the enzyme through the gel. Therefore, the gel was removed after 12 hr of elec-

20l-

IO

7

A 0’

15-

,/’

/

IO-

,

5-

-

/ /’

/I : : :

7 I

L.

I,

-2

I m

I

40

Tube

Fig. 2. Elution

-6

/’

-4

J 0

/’

pattern

I

6C

800

number

of thyroid

xanthine

oxidase

from

LKB electrofocusing column using pH 5-8 ampholine carrier ampholytes.

HYO-SALEE and ALLANG. FISC~IER

564

Table 2. Apparent Michaelis constants (K,) of thyroidal xanthine oxidase Substrate

Acceptor

Xanthine

Oxygen Dichloroindophenol (40 ,uM) Cytochrome c (20 PM) Phenazinemethosulfate (50pM) Nitro blue tetrazolium (40pM) Oxygen Dichloroindophenol (40 PM) Dichloroindophenol (40 yMf Dichioroindophenol (4O~M) Dichloroindophenol (40 PM)

Hypoxanthine NADH NADPH Acetaldehyde

&, (M) 1.6 x 2.8 x 1.7 x 2.5 x 6.7 x 3.4 x 4.3 x 1.2 x 6.1 x 3.2 x

!0-h lo-’ IO-h lo.-6 IO-’ l0-h lo-” lo-* 1o-4 10-l

All values except acetaldehyde were determined with 1OOpg of thyroid xanthine oxidase purified through the G-200 column step. When acetaldehyde was the substrate. the enzyme concentration was increased S-fold.

Table 3. Effect of various additives on thyroid xanthine oxidase activity Concentration

Inhibition

Addition

(mM)

1”J

p-Hydroxymercuribenzoate

0.002 0.005 0.15 0.30 0.65 I.0 5.0 1.0 5.0 I.0 1.33 I.0 I.0 0.5 0.2 0.5 0.5 1.o 1.0 I.0

50 100 73 66 72 0 3x 0 40 0 16 0 0 80 50 96 90 0 0 40

N-ethylmaleimide Sodium arsenite lodoacetic acid Tapazole Thiourea Parnate FDTA Diethyldithiocarbamate Neocuproine Cupferron 2:2’-Bipyridine Sodium azide Sodium cyanide Potassium thiocyanate

Xanthine (66~1M) was used as the substrate and oxygen as the electron acceptor. Same enzyme preparation as in Table 2.

and the enzyme eluted from the gel after maceration. A wide range of molecular weights for xanthine oxidase obtained from several sources has been reported. The iargest difference in molecular weight appears to be associated with the pig liver enzyme. Brumby & Massey (1963) have estimated 385.000 by the Archibald method and 190,000 by gel filtration. The minimum molecular weight of intestinal xanthine oxidase was reported to be 236,000 + 82,000 (Roussos,, 1967). Milk and thyroid xanthine oxidase were run simuftaneously in all of our experiments and we obtained approximate molecular weights of 2~,~ by gel filtration on Sephadex G-200 and 280,000 by disc gel electrophoresis for both enzymes. This is the first report concerning the electrofocusing of xanthine oxidase. An isoelectric point of 6.2 was indicated for the thyroidal enzyme. This value coincides with the isoelectric point of 6.2 for milk xanthine oxidase as determined by a cataphoresis extrophoresis

periment (31). Avis et af. (1956) found the isoelectric point to be between 5.3 and 5.4 by measuring electrophoretic mobilities at different pH values in an acetate buffer of an ionic strength of 0.2. Xanthine oxidase isolated from bovine thyroid glands conforms to the pattern established for the enzyme isolated from other tissues, in that it exhibits a low specificity for substrate and electron acceptors (Table 2). The thyroid enzyme purified as described appears to be an oxidase and has no dehydrogenase activity as NAD+ will not accept electrons from xanthine or hypoxanthine. The rate of xanthine oxidation with oxygen and cytochrome c was about the same magnitude, of the order of 0.1 pmole/min per mg of enzyme. The rate of xanthine oxidation with the artificial electron acceptors, dichloroindophenol, phenazink methosulfate and nitro blue tetrazolium is diminished in comparison to oxygen or cytochrome c as the electron acceptor. Neither of the pyridine nucieotides, NADH or NADPH, nor acetaidehyde

Bovine thyroidal xanthine oxidase was oxidized by molecular oxygen as found for the bovine small intestine xanthine oxidase (Roussos, 1967). However, we observe activity with dichloroindophenol while Roussos (1967) could not with the intestinal enzyme. The milk enzyme is able to oxidize NADH or acetaldehyde with oxygen as the acceptor (Machler et al., 1954). With dichloroindophenol as the electron acceptor and at the indicated saturation levels of substrate, the ratios of the velocities for the oxidation by the enzyme of xanthine (2 x IO-’ M), hypoxanthine (2 x 10e5 M), NADH (20 x 10e5 ML NADPH (20 x 10e5 M) and acetaldehyde (10,000 x 10ms M) are 1.00:0.69:0.54:0.29:0.15. The thyroid enzyme has less activity with acetaldehyde than the intestinal (Roussos, 1967) or milk (Machler et al., 1954) xanthine oxidase. A wide range of K, values has been reported for xanthine oxidase. The K, of 1.6 x 10e6 M with xanthine for the thyroidal enzyme is of the same magnitude reported by Bray (1.0 x 10e6 M) (1959) and Fridovich & Handler (2.65 x 10e6 M) (1958) for the milk enzyme. In contrast, Machler et al. (1954) has reported a K, of 5.5 x 10m5 M for the milk enzyme and Roussos (1967) 2.8 x lo-$ M for the intestinal enzyme. The K, for acetaldehyde of the thyroidal and milk (Machler ef al., 1954) xanthine oxidase is 0.32 M and 0.02 M respectively, with dichloroindophenol as the electron acceptor. In spite of the vast number of publications in the literature, the biological role of xanthine oxidase is not clear. Fried et al. (1973) suggests that the principle role of xanthine oxidase may not be the oxidation of xanthine or hypoxanthine but rather that it serves as a ubiquitous source of hydrogen peroxide. The origin of an in vi00 supply of hydrogen peroxide has long been sought though the requirement of the thyroid gland for hydrogen peroxide, an oxidizing agent which has a sufficient potential (+0.535 v) to oxidize iodide, is well documented (Taurog, 1974). Based on the yield of the pure enzyme, specific activity, K, and V m.l\. it appears that there is a sufficient concentration of xanthine oxidase in the bovine gland to supply the hydrogen peroxide requirement for iodination. Assuming that the K, establishes an approximate value for the intracellular level of substrate (hypoxanthine or xanthine) (Segel, 1975) and accepting the average weight of a thyroid gland (both lobes) to be 50 g (Dinusson, 1949), approximately 600 pmoles of hydrogen peroxide is produced in a 24-hr period. Since the daily requirement of dietary iodide for bovine is 40&8OOpg (3.2-6.3 pmoles), there is an ample supply of hydrogen peroxide being produced by xanthine oxidase to meet the needs of the thyroid gland. However, until control mechanisms are investigated for thyroidal xanthine oxidase, purine nucleoside phosphorylase and deaminase (Dierick er ul., l967), definitive conclusions concerning the importance of nucleoside degradation as a supply of hydrogen peroxide must remain speculative.

565

AVIS P. G.. BERGELF. & BRAYR. C. (1956) Cellular constituents. Chemistry of xanthine oxidase. J. Chem. Sot. 1219-1226. BRAYR. C. (1959) The chemistry of xanthine oxidase. Biothem.

J. 73,

69M94.

BRUMBYP. E. & MASSEYV. (1963) Some properties of xanthine oxygen oxidoreductase isolated from pig liver. Biochem. J. 89, 46. DEGRWT L. J. & CAVALLIE. (1960) Iodide binding in thyroid cellular fractions. J. hiof. Chem. 235, 139c-1397. DIERICKW., OLISLAEGERS P. & S~WKX J. (1967) Adenosine deaminase in bovine thyroid. Archs inf. Physiol. Biochem. 75. 623-634. DINUSSONW. E. (1949) The effects of stilbesterol. testosterone, and thyroid alteration on the growth and fattening of beef cattle. Ph.D. Thesis, Purdue University. Lafayette, Ind. DUKE E. A.. PADDYJ. & RYANJ. P. (1973) Characterization of alternative forms of xanthine oxidase in the mouse. B&hem. 1. 131, 187-190. FISCHERA. G.. SCHULZ A. R. & OLINERL. (I 964) Synthesis of 3:3’-diiodothyronine by beef thyroid microsomes. Biothem. hiophps. Res. CornAn. 14, 39-42. FISCHERA. G.. SCHULZ A. R. & OLINER L. (1967) General characteristics and purification of mitochondrial monoamine oxidase. Biochim. biophys. ACIU 159, 460-471. FISCHER A. G. & LEE H. (1973) Xanthine oxidase from bovine thyroid glands. Life Sci. 12, 267-275. FRIDOVICHI. & HANDLERP. (1958) Xanthine oxidase. J. biol. Chem. 233, 1578-1580. FRIED R., FRIED L. W. & BABIND. R. (1973) Biological role of xanthine oxidase and tetrazolium reductase inhibitor. Eur. J. Biochem. 33, 439445. GORNALLA. G., BARDAWILL C. J. & DAVID M. M. (1949) Determination of serum proteins by means of the biuret reaction. J. biof. Chem. 177, 751-766. HEDRICKJ. L. & SMITHA. J. (1968) Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Archs B&hem. Biophys. 126, 155-164. KANADA M., BRADY F. 0.. RAJAPOPALANK. V. & HANDLERP. (1972) Dissociation of flavin adenine dinucleotide from metallotlavoproteins. J. biol. Chem. 247, 765-770.

KIELLYR. K. (1955) Purification of liver xanthine oxidase. J. biol.

Chem. 216, 405412.

LALANNEM. & WILLEMO~ J. (1975) Xanthine oxidase from mouse skeletal muscle purification and kinetic studies. fnf. J. B&hem. 6, 479-484. LEE H., CARL~ONJ. D.. MCMAHON K. K.. MOYERT. P. & FISCHERA. G. (1977) Xanthine oxidase: a source of hydrogen peroxide in bovine thyroid glands. Life Sci. 20, 453-458. LINEWEAVER H. & BURK D. J. (1934) The determination of enzyme dissociation constants. J. Am. them. SW. 56, 658-666.

LOWRY0. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) J. biol. Chem. 193, 265-275. MACHLERB., MAHLERH. R. & GREEND. E. (1954) Studies on metalloflavoproteins I. Xanthine oxidase. a molybdoflavoprotein. J. biol. Chem. 210, 149-164. MARKLEYH. G., FAILLACEL. A. & MEZEYE. (1973) Xanthine oxidase activity in rat brain. Biochim. biophn Acrtr 309, 23-31. MASSEY V., BRUMBY P.

E.. KOMAIH. & PALMERG. (1969) Studies on milk xanthine oxidase. J. bid. Cbem. 244, 1682-1691.

REFERENCES P. (1964) Estimation of the molecular weights of Proteins by sephadex gel-filtration. Biochm J. 91, 222-233.

ANDREW

MOYERT. P. & FISCHERA. G. (1976) Purification and characterization of a purine-nucleoside phosphorylase from bovine thyroid. Archs Biochem. Biophys. 174, 622-629. ORNSTEIN L. & DAVIS B. J. (1962) Disc electrophoresis. Reprinted by Distillation Products Industries, Rochester, New York.

566

Hvo-SA LEE and ALLAN G. FISCHER

Roussos G. G. (1963) Studies on a hypoxanthine oxidase from bovine small intestine. Biochim. hiophys. Acrcr 73. 338-340. Roussos G. G. (1967) Mrrhon.7 ql’Enz~mo/og!. Vol. 12, pp. 5-16. Academic Press. New York. SEGEL I. H. (1975) Ejqme Kinetics. Wiley, New York. TAURO& A. (1974) Hmdbook of Ph.vsio/ogy. Sect. 7, Vol. 3. p. 101-130. American Phys’iological Society. Washington. D.C.

WHITAKER J. R. (1963) Determination of molecular weights of proteins by gel-filtration on Sephadex. Anulyf. Chetn. 35, 1950-1953. WHITEHEAD J. S.. KAY E., LEW J. Y. & SHANNON L. M. (1971) A preparative column electrophoresis apparatus using Sephadex G-25. Ana/xt. Biochem. 40, 287-291.