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14 J. TANG AND K. I. TANG, J. Bioi. Chem., 238 (1963) 606.15 D. MILLER AND R. K CRANE, Biochim. Biophys, Acta, 52 (1961) 281.16 D. MILLER AND R. K. CRANE, Biochim, Biophys, A eta, 52 (1961) 293.17 R. GITZELMANN, E. A. DAVIDSON AND J. OSINCHAK, Biochim, Biophys, Acta, 85 (1964) 69·18 R. DOELL, G. ROSEN AND N. KRETCHMER, presented to the Society for Pediatric Research,

Seattle, 1964.19 E. EGGERMONT, personal communication (1964).20 A. EICHHOLZ, j. Cell. Bioi., 23 (1964) 27 A.

Received April 7th, 1965

Biochim, Biophys. A eta, 105 (1965) 386-389

sc 63II7Preparation of crystalline i15.3·ketoste roid isomerase from Pseudomonastestosteroni

The enzyme Ll5-3-ketosteroid isomerase (EC 5.3.3.1) which catalyzes the con­version of ,15(6)_ and Ll5(lOL3-ketosteroids to ,14-3-ketosteroids is one of the mostactive catalytic proteins known, and was first crystallized in 1960 (ref. I). Its proper­ties and mechanism of action have been studied2- 4 . For further studies on the struc­ture of the enzyme and its catalytic site, the need arose for larger quantities of theenzyme and for crystals of substantial size. This report describes a simple, large-scalemethod developed for this purpose.

For large-scale growth of induced Pseudomonas testosteroru a 1000-1 fermentorequipped with stirrer and facilities for aeration was charged with 600 1 of a mediumwhich had the following composition per liter of deionized water: I g of (NH4)H2,P04 ;

I g of (NH4)2HP04; 2 g of KH2P04; IO g of Difco yeast extract; 10 ml of trace ele­mentss: and 0.33 ml of Dow-Corning Antifoam C. The sterile medium was maintainedat 30° and inoculated with 61 of a starter culture of Pseudomonas testosteroni (ATCCIIgg6) which had been grown for 16 h with aeration on the same medium. The fer­mentor was aerated at 180 I per min and stirred. After 8.5 h of growth, 180 g of pro­gesterone in about 1.5 1 of acetone were introduced into the fermentor. After z h ofinduction, the specific isomerase activity of cell extracts had increased 49.4-fo1d, andafter 4 h, it had reached 64.S-times the preinduction level. 5 h after addition of pro­gesterone, the tank was cooled to IS° and aeration was discontinued. The bacteriawere harvested with a large Sharples Supercentrifuge at the rate of 1.5-2.0 lJmin.At this rate only 3-5%of the cells were lost in the supernatant fluid. The cell mass wasremoved from the rotor and portions were suspended in 2-1 aliquots of a mediumcomposed of O.OI M sodium-potassium phosphate and 0.001 M EDTA (pH 7-4) bymeans of a Waring blendor (4-1 capacity jar), run at the slowest speed for about 10 sec.The pooled cell suspension (about 24 1) was then passed through a large Sharplescentrifuge in the cold room at the rate of 200 ml/min. The wet cell mass (3.2. kg) wasspread on stainless-steel trays, frozen at -250 and then dried in a vacuum. The driedcell powder was ground finely and stored in a vacuum at _20° (yield 844 g).

The materials used were prepared as follows: Johns Manville Celite AnalyticalFilter-Aid was washed in I-Ib batches on a large Buchner funnel with about 6 1 of0.01 M Tris chloride-so% ethanol (pH 7.0)* followed by 21 of 95% ethanol. The

* The 0.01 M Tris chloride-50% ethanol (pH 7.0) was prepared by mixing equal volumesof 0.02 M Tris chloride of pH 7.0 and 95 % distilled ethanol.

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390 SHORT COMMUNICATIONS

cake was dried at 700. All other preparations and materials have been described-.s.For purificat ion the procedure summarized in Table I was carried out on 400 g

of dried cells .Step 1. Extraction. The finely ground cell-powder was added rap idly to 2400 1111

of 95%ethanol and stirred for I h. The smooth suspension was divided into two equalportions and each received 1200 ml of 0.02 M 'Iris chloride buffer (pH 7.0), and stirringwas continued overni.ght. Each batch of suspension was mixed with 200 g of Celite ,and the thick slurry was poured onto a Buchner funnel (31.5 em diameter) coveredwith a single layer of filter paper and a bed of Celite (75 g) which had been poured

TABLE I

PURIFICATIOror OF Ll'-3-KETOSTEROID ISOMERASE

One uni t of iso merase activi ty causes the isomerization of I pmole of .d'-andlOstene-3, 17-d ioneper min at 25° and pH 7.0, in a reaction syst em of 3.0 ml volume containing: lOO pmoles of potas­sium phosph at e buffer , 0.17 5 .umole of Ll'-an drost ene -3,I7-d ione in 0,05 m l methanol, and enzymeappropriately dilut ed in I % crystalline bovine serum albumin at pH 7.0. Pro tein determinationswer e made by the biuret m ethod , except for the crystalline enzyme, for which the absorbancy at280 mfl was us ed , assuming that I mg of pure isomerase per ml has an A 280 = 0.413 (sec ref. 2).

Volume Protein Total Spec ific Purifi- Yie ld{ml} concen - activity activity cation

tration (units X (units (%){mglml} IO-B) permg

proteinX IO-')

First extract 4560 0 ·44 4.22 2.IISecond extract 1800 0.30 0.410 0.760Tot al ini t ial extract 6 360 0·36 4·33 1.89 100

Calcium phosphate gel eluat e(afte r d ialysis) 360 2.8 5.29 5. 24 2·77 122

Combined DEAE-cellulose efflu ent(Fracti ons ~4, 25 and 26) 29·7 3.96 2·7° 23.0 12 .2 62 ·5

0-70 % (NH4)2S0. 13·4 12.8 2·93 17.1 9.05 67.5Firs t crystals 3.0 21.4 2.63 4° ·9 21.6 61Second cr yst a ls ' 10·7 3.25 3.08 88·5 46.8 71.1

• Dissolved in 2 ,0 m l 0 .005 M potassium p hosp hate (pH 7.1) and passed through a Sepha­dex G-25 colu mn (12.5 X 2.2 em) eq uilibrated with same buffer. The entire activity was recoveredin 10.7 ml on wh ich protein a nd activity determin at ions were made.

in 0.01 M Tri8- 50% ethanol. After comp letion of the filt ration under vacuum whichrequired approx. X h, each cake was washed with 500 ml of O.OI M Tris-50% ethanol.The chartreuse-colored filtrates were pooled (4560 ml). Each filter cake was suspendedin 700 ml of 0.01 M Tris-50 % ethanol by gentle mixing in the 4-1 cup of a Waringblendor operated at reduced speed. The suspension was then stirred with a magn eticbar for one additional hour. Because the mixture was too viscous to filter , it wascentrifuged at IO 000 X g for 30 min . The pale yellow supernatant (1800 ml) wascombined with the pooled filtrates (total volume of 6360 ml).

Step z. Calcium pho sphate gel adsorption and elution. For convenience thepooled extracts were again divided into equal portions which were placed into 2

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SHORT COMMUNICATIONS 391

rz-I bottles. To each aliquot (3180 ml) was added in the following order: 4725 ml of95 % ethanol, 39.4 ml of La M MgCl2 and approx. 20 ml of calcium phosphate gel(r8 mgjml, dry weight). The mixture was stirred for I h. The gel was permitted tosediment overnight. Most of the supernatant fluid could then be removed by gentlesiphoning. The gel was resuspended in the residual fluid (about 1.2 l) and centrifugedfor 15 min at 1500 X g. The supernatant contained negligible activity and wasdiscarded. The gel was washed with 220 ml of 80% ethanol-o.oog M MgCl2-0.01 MTris chloride (pH 7.0). The suspension was centrifuged and the supernatant was dis­carded. The enzyme was then eluted from the gel in two steps. The first elution in­volved stirring for 30 min with ISO ml of 0.01 M potassium phosphate buffer (pH 7.0)followed by centrifugation for IS min at r500 X g and gave 132 ml of orange super­natant fluid. The second elution was carried out with go ml of 0.1 M potassium phos­phate (pH 7.0) in an identical manner and gave 96 ml of supernatant fluid.

Step 3. Dialysis and chromatography. The combined eluates (222 ml) weredialyzed for 24 h against 4 changes of 4 I each of 0.001 M Tris phosphate (pH 7.0).The first three changes were bright yellow whereas the fourth change was almostcolorless. The dialyzed solution (360 ml) was applied to a DEAE-cellulose column(22·3 em X 3.3 em, equivalent to approx. 20 g of dry powder), which had beenpreviously equilibrated against o.oor M Tris phosphate (pH 7.0). Elution was carriedout by a convex gradient obtained from a constant-volume mixing chamber (Soo-mlcapacity) containing o.oor M Tris phosphate (pH 7.0), connected to a reservoir of004 M Tris phosphate (pH 7.0). Fractions (ro ml) collected at a rate of 1.7 mljmin,were numbered from the attachment of the gradient to the column. Most of the activi­ty was contained in Fractions 23-27. Fraction 24 contained about 68% of the totalrecovered activity. The enzyme activity was eluted just ahead of a yellow pigment.

Step 4. Ammonium sulfate precipitation and crystallization. Fractions 24, 25and 26 were combined, and solid ammonium sulfate was added to 70% saturation.The precipitate was allowed to accumulate overnight, collected by centrifugation anddissolved in a small volume of 0.03 M potassium phosphate buffer (pH 7.0). The slight­ly turbid solution (1304 ml) was clarified by centrifugation, and a saturated solutionof twice recrystallized ammonium sulfate (adjusted to pH 7.0 with NH40H) wasadded drop-wise until permanent turbidity was observed. The solution rapidly becamegelatinous in consistency, and 4.8 h later, the solution had the silky sheen upon swirl­ing, characteristic of crystalline enzyme. The crystals were dissolved in 0.03 M potas­sium phosphate buffer (pH 7.0) and recrystallized by addition of saturated neutralammonium sulfate solution.

Pure isomerase crystallizes without difficulty in uniform needles or elongatedplatelets- within a few minutes to several hours after the addition of ammoniumsulfate to incipient turbidity, provided the protein concentration does not exceedIO-I2 mgjml, The size and uniformity of the crystals obtained depends upon theirrate of formation. Uniform large crystals easily visible with the naked eye (1-2 mmlong) were obtained in 7-14 days at 25° when the ammonium sulfate concentrationwas 26.1% of saturation, and the protein concentration was about 4.5 mgjml. Thesame solution at 4° crystallized only after several months. If the ammonium sulfateconcentration was between 37.5 and 28.6% of saturation, microscopic crystals wereobtained within r h or less at 25°. The size of the crystals was smallest when thecrystals formed rapidly.

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392 SHORT COMMUNICATIONS

These studies were supported by a grant from the United States Public HealthService (AM 07422).

Department of Pharmacology andExperimental Therapeutics,The Johns Hopkins University School of Medicine,Baltimore, Md. (U.S.A.)

PAUL TALALAY

JEAN BOYER·

I F. S. KAWAHARA AND P. TALALAY, J. Bioi. Chem., 235 (t960) PC I.

2 F. S. KAWAHARA, S.-F. 'WANG AND P. TA(..ALAV, J. Bioi. Chem., 237 (1962) 1500.3 S.-F. WANG, F. S. KAWAHARA AND P. TALALAV, J. Bioi. Chem., 238 (1963) 576.4 P. TALALAV, Ann. Rev. Biochem., 34 (1965) 34-7·5 F. S. KAWAHARA, in S. P. COLO WICK AND N. O. KAPLAN, Methods i1t Enzymology, Vol. V,

Academic Press, New York, 1962, p. 527.

Received April 7th, Ig65

• Permanent address: Clinique d'Endocrinologie de la Faculte de Medecine de Marseille,Marseille (France).

Biochim; Biophys, Acta, 105 (1965) 389-392