ARCHIVES

OF BIOCHEMISTRY

Purification

AND

174, 622-629

(1976)

and Characterization of a Purine-Nucleoside Phosphorylase from Bovine Thyroid’

THOMAS Biochemistry

BIOPHYSICS

Department,

P. MOYER” North

AND

ALLAN

G. FISCHER:’

Dakota

State

Uniuersity,

Fargo,

Received

July

10, 1975

North

Dakota

58102

A purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) from bovine thyroid tissue has been purified 670-fold utilizing the techniques of ammonium sulfate precipitation, ion-exchange and molecular-exclusion chromatography, and polyacrylamide-gel electrophoresis. The protein has an apparent molecular weight of 90,000, a single isoelectric point at 5.6, and a Michaelis constant of 0.028 mM for inosine. Double-reciprocal plots of the reaction rate for the phosphorylasecatalyzed reaction versus phosphate or arsenate concentration display a downward trend at high substrate concentrations. Two apparent Michaelis constants of 0.38 and 1.49 mM were determined for phosphate.

This laboratory has previously reported the identification of the enzyme xanthine oxidase (EC 1.2.3.2) from bovine thyroid tissue (1). Evidence was offered (2, 3) to substantiate that this enzyme may act as a source of hydrogen peroxide necessary for the oxidation of iodide in the biosynthesis of the hormone thyroxine. A study for the source of substrates for xanthine oxidase in the thyroid gland reveals the existence of a purine-nucleoside phosphorylase in the soluble fraction of a thyroid homogenate. This paper deals with the isolation procedure and characterization of the thyroidal PNPase.’ A PNPase has not been previously isolated from thyroid tissue. Parks and Agarwal (4) speculate that the enzyme may be involved in both synthesis and breakdown of nucleosides. However, in mammalian tissue the PNPase functions primarily in degradation, There ’ Journal Article Number 626, North Dakota Agricultural Experiment Station. 2 Present address: Department of Laboratory Medicine, Mayo Clinic, Rochester, Minn. 55901. :I To whom reprint requests should be sent. purine-nucleoside ’ Abbreviations: PNPase, phosphorylase; DTE, dithioerythritol; PPAGE, preparative polyacrylamide-gel electrophoresis; bisTris, bis-(2-hydroxymethyl)-imino-tris-(hydroxymethyljmethane; DEAE-, diethyl aminoethyl. 622 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

are two manifestations of the enzyme: (i) It may act as an orthophosphate:ribosyltransferase, causing the breakdown of one nucleoside with the concurrent formation of another nucleoside, or (ii) it may act as a nucleoside phosphorylase, causing the breakdown of a purine nucleoside, with the product ribose l-phosphate being incorporated via phosphoribomutase and 5-phosphoribosylll-pyrophosphate synthetase into 5phosphoribosyl/l-pyrophosphate. In either case, the PNPase acts in a salvage capacity. A preliminary report of the following work has been presented (5). MATERIALS

AND

METHODS

Purification Bovine thyroid glands were obtained from a local abattoir within 15 min of slaughter. Glands (0.5 kg), free of connective tissue, were homogenized in (1.1 liters) 0.2 M bicarbonate, pH 7.7, 0.1 mM DTE, and 1 mM ethylenediaminetetraacetate in a Lourdes homogenizer (Model MM-1B) for 2 min, followed by centrifugation at 45,OOOg for 1 h. The soluble fraction of the homogenate was fractionated by addition of a 20%’ volume of n-butanol followed by centrifugation at lO.OOOi: for 10 min. The aqueous supernatant of the butanol centrifugate was subjected to ammonium sulfate fractionation, with the protein precipitated between 30 and 55% ammonium sulfate saturation showing phosphorylase activity. The precipi-

THYROIDAL

PIJRINE-NUCLEOSIDE

tated protein was resuspended in 50 ml of water and dialyzed against 80 volumes of 0.01 M phosphate, pH 6.2, 0.1 mM DTE before application to a 2 x 60-cm column of DEAE-cellulose equilibrated in 0.01 M phosphate, pH 6.2, 0.1 mM DTE. Elution of the enzyme was performed by increasing phosphate concentration in a linear gradient formed from 300 ml of 0.01 M and 300 ml of 0.2 M phosphate, pH 6.2, containing 0.1 mM DTE. Fractions containing phosphorylase activity, eluted in the last half of the gradient, were pooled and the volume reduced by ultrafiltration tAmicon ultrafiltration chamber, PM-10 membrane) to 5 ml. Phosphorylase activity was separated from the smaller molecular weight proteins by a 4.5 x 60.cm Sephadex G-100 column equilibrated in 0.1 M phosphate, pH 6.2, 0.1 mM DTE. The exclusion eluate containing enzymic activity was dialyzed against 40 volumes of 0.1 mM DTE for 8 h and treated with calcium phosphate gel (14 mgimli, prepared in the manner of Tsuboi and Hudson (6), added in a ratio of 1 ml of gel per 2.4 mg of protein. The suspension was centrifuged and the residues combined by suspending each pellet in approximately 5 ml of 0.1 mM DTE. The gel was washed once with 40 ml of0.1 rnM DTE and twice with 40 ml of 0.01 M phosphate, pH 6.2, 0.1 mM DTE. The phosphorylase activity was eluted by three 50-ml washings with 0.1 M phosphate, pH 7.0, 0.1 mM DTE. The washings were combined and concentrated by ultrafiltration. Further purification was performed on a Buchler preparative polyacrylamidcgel elcctrophoresis apparatus with a chemically polymerized separating gel (15%) at pH 8.9 and a photopolymerized concentrating gel (2.5%) at pH 7.2. The elcctrophoresis buffer system consisted of 0.1 M Tris-HCl, pH 8.1, 0.1 mM DTE as a lower buffer and 0.053 M Tris-glycine, pH 8.9, 3 mM thioglycolate, as an upper buffer (7). Electrophoresis at a constant current of 50 mA for 24 h with continuous elution by the lower buffer achieved partial separation of the proteins. Analytical gel electrophoresis was performed in a Buchler Polyanalyst apparatus for 1 h using the same system of gels. The analytical buffer system consisted of 0.053 M Tris-glycine, pH 8.9, as the upper buffer and 0.1 M Tris-HCl, pH 8.1, as the lower buffer, Isoelectric focusing of the enzyme was carried out m the manner of Vesterbcrg and Svenssen (8) in an LKB 8102 Ampholine column. A I’?r ampholite concentration containing 0.1 mM DTE in a sucrose density gradient was used as the column medium. Power was maintained at 3 W until the voltage reached 1000 V. At a constant voltage of 100 V, the column was charged until the power reached a constant value of 1.2 W (48 hi. All purification procedures were performed at I)-5°C. Molecular weight determinations were performed by the method of Whitaker 19) utilizing a 1.2 x 48. cm column of Sephadex G-200, equilibrated in 0.1 M

PHOSPHORYLASE

623

phosphate, pH 7.0, 0.1 mM DTE, 3 mM sodium azide. Proteins of known molecular weight used as column alcohol dehydrogenase were yeast standards beef heart lactate dehydrogenase (150,000~, (140,000), soybean lipoxygenase (102,000), horse liver alcohol dehydrogenase (75,000), and human hemoglobin (64,000). Distribution coefficients iv,./ V,,) were determined by assaying individual enzymes, using dextran blue to calculate the void volume (V,,).

Assays Enzyme assays were performed using a recording Pcrkin-Elmer 124 spectrophotometer with temperature control. The action of the enzyme on guanosine was measured by a change in absorbance at 257 nm (4). A coupled enzyme assay utilizing milk xanthine oxidase as the second enzyme was used to measure enzyme activity on inosine or xanthosine by the method of Kalckar (10~. The product, uric acid, has an optimum absorbance at 292 nm. The standard inosine assay contained 0.3 mM inosine in 0.06 M phosphate, pH 7.0, and 0.01 unit of milk xanthine oxidase in a volume of 3.0 ml. Phosphorylase activity was adjusted to assure less than 0.0025 unit per assay in a total volume of 3.1 ml. The release of ribose ias ribose l-phosphate1 from purinc nucleosides was measured by reaction with orcinol (111. Enzyme and substrate (20-200 rnM) were incubated for specified times (I-5 mini in 0.06 M phosphate. pH 7.0. The reaction was stopped by addition of 10% trichloroacetic acid containing 10% activated carbon. Centrifugation removed all traces of the protein and substrate. The supernatant was treated with an acidic orcinol solution and assayed for pentose content by monitoring the absorbance at 670 nm. Ribosyltransferase activ-ity was assayed in the manner outlined by Kimet al. (12). A volume of 3.15 ml contained 0.42 mM inosine, 0.42 mM guanine, xanthinr 0.06 M Tris, pH 7.1, 0.01 unit of milk oxidase, and 0.0025 unit of PNPase. All assays were performed at 30°C. A unit of activity is defined as 1 pmol of substrate converted to product per minute. Protein concentrations were measured by the method of Lowry et al. (1.31 for low concentrations, and the biuret method (14) was used for high conccntrations. The protein content of column chromatography cluates was measured by a change in absorbance at 280 nm.

Chemicals Trizma base, bis-Tris, DTE, ammonium sulfate, sodium thioglycolatc, milk xanthine oxidase, purine bases and nuclcosides, and all molecular weight standards were obtained from Sigma Chemical Co. Sephadex was from Pharmacia Fine Chemicals.

624

MOYER

AND

and triphenyl tetrazolium Acrylamide, orcinol, chloride were obtained from Eastman Chemical Co., and ampholites were from LKB Instrumentation, Inc. All reagents were reagent grade. RESULTS

A purine-nucleoside phosphorylase was isolated from the soluble fraction of a bovine thyroid cellular homogenate. An aliquot of the homogenate, when centrifuged in a Beckman Model L ultracentrifuge at 105,OOOg for 1 h, gave a clear supernatant containing 100% of the activity found in the initial 45,000g supernatant. Table I outlines the stage of purification achieved by each step described in the Materials and Methods for one typical isolation. The average enzyme activity of the crude homogenate from 12 different thyroid collections was determined to be 0.59 t 0.09 unit/g of whole tissue. To ensure that the isolated phosphorylase activity was from thyroid tissue and not from erythrocytes (a common source of PNPase), whole bovine blood was treated in a manner identical to the thyroid tissue. The activity from the whole blood homogenate was determined to be 0.037 unit/ml of whole blood. Since the thyroidal homogenate specific activity was 16 times that of whole blood, the contaminating PNPase from erythrocytes was considered to be negligible. Isoelectric

pH

Adsorption of thyroidal PNPase by DEAE-cellulose and calcium phosphate at pH 6.2 indicated that the enzyme has an TABLE PURIFICATION

Fraction

Total ume

Homogenate Butanol extract (NH,),%), DEAE-cellulose Sephadex G-100 Calcium-PO, PPAGE ” Activity determined utilizing Ir Expressed as units/milligram

DATA

FOR PURINE

vol(ml)

Total activity (units)

FISCHER

anionic character at that pH. This fact was substantiated by the determination of the isoelectric point, indicated in Fig. 1. The localization of PNPase in a gradient of pH 4 to 9 indicated that the isoelectric point of PNPase is 5.6. The process of isoelectric focusing resulted in partial deactivation of the enzyme. Only 10% of the enzymic activity was recovered after 48 h of focusing at 4°C in the presence of reducing agent. Molecular

Weight Estimation

The molecular weight of the PNPase was estimated to be 90,000, assuming spherical symmetry, using Sephadex G200. Five determinations were performed with a variation in distribution coefficient calculated to be less than 3% for any of the protein standards. Acrylamide

Electrophoresis

Figure 2 records the elution pattern of PNPase from the PPAGE system. The protein migrates through the 15% gel within 24 h. In the absence of reducing agent, only 10% of the enzymatic activity introduced onto the column could be recovered. When 3 mM thioglycolate was added to the upper buffer, recovery was greatly increased. Analytical gel electrophoresis of the PPAGE eluant (Fig. 3) followed by protein staining with aniline blue black showed one major band and four minor bands. Immediately after electrophoresis, a gel was treated with tetrazolium chloride by the method of Mattson and Jenson (15). The PNPase was shown to be associated with the one major protein band. SubstituI NUCLEOSIDE

Specific

PHOSPHORYLASE

activity”

1260 1340

371.8 331.6

0.00678 0.00686

101 363 187 146 169

190.2 142.5 142.4 83.7 70.7

0.194 0.817 1.52 1.79 4.55

the standard of protein.

inosine

assay.

Recovery (?;I

Purification (n-fold)

100 89

1 1.02

51 38 38 23 19

18.7 120 224 264 671 _____ ~

THYROIDAL

PURINE-NUCLEOSIDE

4. 10

20

xl

40

Tube Number

625

PHOSPHORYLASE

50

70

60

I

(6ml/tube)

1, Isoelectric focusing of the PNPase purified 670-fold by PPAGE was Ampholine 8102 focusing column containing a 1% ampholite solution, 0.1 mM of PNPasr in a sucrose density gradient. The pH (O---O) was measured on 110 pH meter The PNPase activity CO---0) was measured by the standard FIG.

20

40

60

Tube Nmber FIG.

solid K-O)

lint

80

03

Enzyme Stabilization Reducing agents were required to maintain enzymic activity. Dithioerythritol (0.1

140

(15 ml/lube)

2. Elution pattern of preparative polyacrylamide-gel represents the UY absorption of the column eluate was measured by the standard inosinc assay.

tion of the 15% gel with a 7.5% gel in the analytical system changed the respective mobilities, but staining with aniline blue black still revealed five bands, one of them considerably more intense than the others. The analytical gel effectively separated the proteins, while in the preparative system there was much overlapping of the protein bands as evidenced by the impurities which are indicated in the analytical gel (Fig. 3). This electrophoretic behavior was consistent in four separate isolations.

120

performed on an DTE and 20 units a Corning Model inosine assay.

electrophoresis at 280 nm. The

(PPAGE). The PNPase activity

mM) was found to be effective in stabilizing the PNPase in a buffered system. After 9 h of dialysis of the PPAGE preparation against three changes (3 h each) of 20 volumes of 0.1 M phosphate, pH 7.0, 5% of the activity was lost. Further loss of activity was noted upon storage of the enzyme at 4°C. Six hours after the completion of dialysis, 82%’ of the initial activity remained, and at the end of 72 h no activity could be detected. The activity could not be restored by incubating the enzyme at 4°C in 0.1 M phosphate, pH 7.0, 0.1 mM DTE for 24 h. Other reducing agents, 0.1 mM dithiothreitol, 10 mM mercaptoethanol, and 3 mM thioglycolate, were effective in

626

a A

B

,i

PPAGE IEF AniliM BkJe

MOYER

AND

C

:

PPAGE T$+ypm

8

@

Block Soin FIG. 3. Analytical polyacrylamide gels of purified PNPase. Protein applied to gels A and C was from the PPAGE purification step. Gel B represents protein after isoelectric focusing (IEF). Gels A and B were stained with aniline blue black followed by destaining in 7% acetic acid. Gel C was incubated in 0.06 M KH,PO,, pH 7.0, 0.30 mM inosine at 30°C for 15 min. Following incubation, the liquid was drained off and equal volumes of 1 N NaOH and 0.5% 2,3,5-triphenyl-2H-tetrazolium chloride were added. A brown stain on the gel indicates the presence of PNPase.

FISCHER

tration 0.1 M) to the enzyme incubation medium initiated a change in absorbance at 292 nm. Incubation of the mixture containing phosphate for 20 min at 30°C followed by the orcinol treatment indicated the presence of ribose in the system. Replacement of phosphate by arsenate also initiated a change in absorbance at 292 nm. Incubation of 0.15 mM guanine, 0.05 mM ribose l-phosphate, 0.06 M phosphate, pH 7.0, with 0.0025 unit of the enzyme produced an increase in absorbance at 257 nm. When 0.05 mM ribose was substituted for ribose l-phosphate in the reaction no change in absorbance at 257 nm was observed. Incubation of inosine with PNPase and xanthine oxidase in the absence of phosphate displayed no change in absorbance at 292 nm. Addition of 0.25 pmol of guanine resulted in no change at 292 nm. When the initial incubation mixture (less guanine) included 0.25 Fmol of phosphate, the change in absorbance at 292 nm was noted to be 0.0039 2 O.O006/min (n = 4). Addition of 0.25 pmol of guanine to the reaction caused a decrease to 0.0023 t O.O003/min (n = 4). The variability of the results was confirmed by repetition of n assays,

maintaining enzymatic activity. The absence of buffering salts also resulted in loss of enzyme activity. Dialysis of the PPAGE-purified protein preparation against four changes of 20 volumes of 0.1 mM DTE at 4°C (12 h for each change) resulted in 85% inactivation of the phos- Substrate Specificity The enzyme was maximally active with phorylase activity. Dialysis against 1.0 of mM phosphate, pH 7.0, 0.1 mM DTE for an inosine at pH 7.0 (Fig. 4). Substitution additional 24 h increased the activity to xanthosine for inosine changed the pH optimum to 5.2. The ratio of enzyme activi20% of the original preparation. Increasing the concentration of phosphate to 0.1 M ties in the degradation of inosine and xanand dialysis for 8 h at 4°C restored 100% of thosine at pH 7.0 and 5.2, respectively, the original activity. Repetition of the ex- remained the same with enzyme samples from the crude homogenate, ammonium periment, replacing the phosphate buffer sulfate precipitate, PPAGE, and isoelecby 0.1 M bis-Tris, pH 7.0, gave similar tric focusing. The substrates guanosine results. and adenosine, when acted upon by the Identification of Enzymatic Activity partially purified PNPase (void of deamishowed optimal activity at Incubation of the enzyme in 0.3 mM ino- nase activity), pH 7.0, but with lower rates. sine, 0.1 M Tris, pH 7.2, 0.01 unit of milk xanthine oxidase, and 0.0025 unit of thyKinetic Parameters roidal PNPase (specific activity 4.5 units/ The Michaelis constant (K,) was determg) produced no change in absorbance at and 292 nm. After incubation for 20 min, no mined by the method of Lineweaver ribose could be detected by the orcinol pro- Burk (16) for the substrate inosine (Fig. 5) to be 0.028 mM. Other substrates are listed cedure as outlined in Materials and Methods. Addition of phosphate (final concenin Table II. Inhibition of PNPase by both

THYROIDAL

PURINE-NUCLEOSIDE

627

PHOSPHORYLASE

PH

FIG. 4. pH profile, The PNPase (0.2 unit/mg, activity was measured Tris-phosphate buffer from pH 4 to 8 and in 0.1 M phosphate-Tris-glycine 10 using the orcinol assay to measure reaction rates. Substrates indicated xanthosine (O-O), guanosine (O--O), and adenosine (V---Vi.

III 0.1 M acetnte-bisbuffer from pH 7 to are inosine (V--Vi,

FIG. 5. Double-reciprocal plot of PNPase (4.5 units/mg) activity with varying inosine concentration. Substrates indicated are inosine (O-O), inosine plus 0.01 mM guanine (G-91, and inosine plus 0.04 mM adenine (V---O). Reaction rates were determined by varying concen trations of inosine in 0.06 M KH,PO,, pH 7.0, 0.01 unit of milk xanthine oxidase and 0.0025 unit of PNPase and monitoring the change in absorbance at 292 nm

guanine and adenine, when the substrate was inosine, is also indicated in Fig. 5. Indicated in Fig. 6 are the effects of varied concentration of the substrates phosphate (Fig. 6a) and arsenate (Fig. 6b) on the reaction rates. In either case a nonlinear relationship was noted. Two values of the Michaelis constant may be determined. For phosphate, they are 0.35 and 1.49 IIIM. For arsenate, they are 0.84 and 1.96 mM. Similar values were obtained when the second substrate was either inosine or guanosine.

DISCUSSION

An enzyme isolated from bovine thyroid tissue shows evidence of being a purinenucleoside phosphorylase. The phosphate and arsenate dependence, the studies on the reverse reaction, and the fact, that ribose is not cleaved from a nucleoside until phosphate ribose

l-phosphate

+ guanine $ guanosine

+ P,

is added indicate that the react.ion is phosphorylytic and not hydrolytic.

628

MOYER TARLE

KINETIC

II

CONSTANTS FOR PURINE PHOSPHORYLASE

Substrate Inosine” Xanthusine” Guanosine” Adenosine’ Deoxyinosine” Deoxyguanosine” Deoxyadenosine’

AND

Km (PM) 28 95 22 130 27 21 145

NUCLEOSIDE

V” (units) 49.5 42.0 35.8 18.1 48.0 31.2 17.0

” Activity measured in 0.06 M KH,PO,, pH 7.0 txanthosine measured in 0.05 M KH,PO,, 0.05 M KC,H,,O,. pH 5.21, containing varying substrate concentrations. Milk xanthine oxidase (0.01 unit) was added with 0.0025 unit of PNPase to a total volume of 3.1 ml. Activity noted as a change in absorbance at 292 nm. ” Activity measured in 0.06 M KH,PO,, pH 7.0, varying substrate concentrations, and 0.025 unit of PNPasr. hctivity noted as change in absorbance at 251 nm. substrate concentrations in 0.06 M ’ Varying KH?PO,. pH 7.0, with 0.0025 unit of PNPase. Ribose formation at 1 and 2 min measured by the orcinol method allowed rate determinations. Specific activity of the PNPase used in these studies was 4.5 umtsimg ,I V represents maximum velocity.

The thyroidal PNPase has an apparent molecular weight of 90,000. In general, the molecular weights of PNPases from mammalian tissue have tended to range from 79,000 to 83,000 (4). The molecular weight of thyroidal PNPase could vary from 88,500 to 91,500, suggesting that the enzyme may be different from other mammalian PNPases. The low K,,, and high V (Table II) expressed by the enzyme with inosine and guanosine may indicate that these nucleosides are the natural substrates. The pH optimum at 5.2 with xanthosine is atypical when compared with the other substrates tested (Fig. 4) and a complete lack of activity at the pH where the other substrates show maximal activity tend to minimize the physiological significance of this particular enzymatic activity. The spectrophotometric assay for ribosyltransferase activity repeatedly showed the change in absorbance at 292 nm to be zero, signifying a lack of this activity associated with the 670-fold-purified PNPase.

FISCHER

Dialysis of enzyme preparations against phosphate-free buffers was found to be adequate in the elimination of phosphorylase activity. Inhibition of PNPase activity by either guanine or adenine with inosine as substrate (Fig. 5) is offered as additional evidence against a ribosyltransfer reaction. Activation, as would be expected for a pentosyl transfer reaction, was not observed. Further, when a phosphorylase reaction was initiated by incorporation of 0.25 Fmol of phosphate into a phosphatefree system, followed by addition of guanine, there was a decrease in reaction rate. These conditions should establish an apparent ribosyltransfer reaction, as defined by Kim et al. (12). The observed decrease in reaction rate implies that there was no ribosyltransfer activity. Agarwal et al. (17) were able to identify two or more isoelectric variants from human and rat erythrocyte PNPase. Figure 1 indicates a single peak. This is similar to the bovine spleen enzyme, which was shown to give a single variant (17). The ratio of activities with inosine at pH 5.2 remains constant in the fractions from the isoelectric focusing experiment. The same was true for samples taken from each step of the purification scheme, leading to the conclusion that this activity is expressed by one enzyme or isozymes that are inseparable by p1 using the outlined method. Figure 6 shows the interesting phenomenon of a downward trend occurring in the double-reciprocal plot indicating enzymatic activation occurring at high substrate concentration. Kim et al. (12) and Agarwal et al. (17) have reported a similar phenomenon to occur with the purine substrates using enzyme purified from human erythrocytes. They report this behavior is due to substrate activation rather than the existence of isozymes. However, activation of a PNPase by phosphate has not been previously reported. The cause of the activation of the thyroidal PNPase cannot be determined at this point due to the lack of homogeneity of the enzyme preparation. ACKNOWLEDGMENT Appreciation is expressed to Flavorland Food dustries, West Fargo, N. D., for their contribution a generous supply of bovine thyroid glands.

Inof

THYROIDAL

PURINE-NUCLEOSIDE

629

PHOSPHORYLASE

a 200 -

I 05

IO

15

20

25

30 I/S

05

IO

15

20

25

30

(mM)-’

FIG. 6. Double-reciprocal plots of PNPase (4.5 unitsimg) activity with varying phosphate (a) or arsenate (b) concentrations. Reaction rates were determined by varying phosphate or arsenate concentrations in 0.06 M Tris, pH 7.1, 0.3 rnM inosine, 0.01 umt of’ milk xanthinr oxidase, and 0.0025 unit of PNPase and monitoring the change in absorbance at 292 nm.

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A. G.. AND

267-275. H. (19741 State University,

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YAMAMOTO,

K.,

LEE,

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H. (1973)

Lifi

Sci.

12. 10.

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DEGROOT,

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L. J. (1975)

En-

tloc~rinolog~v 96, 1022-1029. 4. PARKS, R. E.. AND AGARWAL, R. P. (1972) in. The Enzymes (Bayer, P. D., ed.). Vol. 7. pp. 483514, Academic Press, New York. 5. FISCHER, A. G., AND MOYER, T. P. (1974) Abstracts of Papers, 168th American Chemical Society Meeting, B-136. 6. TSUBOI, K. K., AND HUDSON, P. B. (195715. Biol. Chem. 223, 879-887. 7. MAURER, H. R. (19711 Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, pp. 41-45, Walter de Gruyter. New York. 8. VESTERBERG, O., AND SVENSSON, H. (1966) Ada Chcm. Scnnd. 20. 820-834.

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(1963)

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35,

l!%O-

C/?rzrn.

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1953. KALCKAR,

H. M.

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443. 11. DEWEY, V. C.. AND Rzochem Biophys.

KIDDER,

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119731 Arch.

G. W.

1.57, 380-387. R. E. (1968)

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0. H.. R~SEBROU~H, N. J., F’ARR. A. L.. R. .J. I 1951 1.1. BII)(. (‘htar,~. 193,

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265-275. 14. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M. (1949) ./. Blol. C’hc,nr. 177, 751-766. 15. MATTSON. A. M.. ANDJENSON. C. 0. (1950iAnul. Chem. 22, 182%18s. 16. LINEWEAVER, Chem. Sot.

H.. AND BURK, 56, 658-666.

17. AGARWAL, J. 0..

PARKS,

K. C.,

AND 11, 79-84.

AGARWAL,

D. I 1934) ./. ilmcr. R. P.,

STOECKLER.

R. E. (19751 Nrochcrnlstr,>