SHORT COMMUNICATIONS 151

BBA 6319 ° Isolation of 3-oxosteroid At_AS -isomerase of human term placenta

In a previous s tudy , evidence was presented t h a t the 3- f l -hydroxys tero id : NAD(P) oxidoreductase (EC 1.1.1.51) and the 3-oxosteroid A4-AS-isomerase (EC 5.3.3.1) ob ta ined from h u m a n t e rm p lacen ta might be separa te enzymes 1. I t was also suggested t ha t the isomerase ac t i v i t y might be composed of mul t ip le enzymes. In this s t u d y the p lacen ta l homogena te was t r ea ted wi th acetone and deoxycho la te to solubil ize the isomerase a c t i v i t y and f rac t iona ted subsequen t ly wi th a m m o n i u m sulfate.

In i t i a l a t t e m p t s to ob ta in the enzyme from the subcel lular f ract ions were un- successful. F o r this reason the ent ire p lacen ta was used. Acetone powder of h u m a n t e r m p lacen ta was p repa red in the usual manner . The powder was homogenized wi th o.15 M KC1 (i g/2o ml) in a W a r i n g b lendor a t full speed for I min. The homogena te was s t i r red for I h a t 0-2 ° and centr i fuged at 20 ooo × g for 20 min. The supe rna t an t solut ion was placed in a Visking casing and d ia lyzed overnight aga ins t several changes of 1. 5 M (NHa)2SO 4 ad jus t ed to p H 7.0 wi th d i lu te NH4OH. The re tente was centr i - fuged at 20 ooo × g for IO min. The sed iment was dissolved in o .o i M phospha te buffer and centr i fuged at 30 ooo x g for 20 minutes . The sed iment was resuspended in o .o i M phospha te buffer (pH 7.0). The supe rna t an t solut ion f rom the 1.5 M (NHa)2SO a f ract ion was placed in a Visking casing and d ia lyzed overnight aga ins t several changes of 3.0 M (NH4)2SO 4 ad jus t ed to p H 7.0 wi th d i lu te N H a 0 H and cen- t r i fuged at 20 ooo × g for IO rain. The sed iment was dissolved in o .o i M phospha te buffer, (pH 7.0) d ia lyzed overn ight aga ins t severa l changes of o .oi M phospha te buffer, (pH 7.0) and centr i fuged at 30 ooo × g for 20 min. The sed iment was resus- pended in o .0 i M phospha te buffer (pH 7.0).

As an a l t e rna t ive method , fresh h u m a n t e rm p lacen ta was homogenized wi th a m e d i u m conta in ing 0.003 M sod ium deoxychola te , o . i M po tas s ium phospha te buffer (pH 7.0) (I g/3 ml) in a War ing b lendor at full speed for I rain. The homo-

TABLE I

3-OXOSTEROID Z]4--~5-ISOMERASE ACTIVITIES WITH 5 - P R E G N E N E - 3 , 2 0 - D I O N E AND 5 - A N D R O S T E N E -

3 , I 7 - D I O N E IN AMMONIUM SULFATE FRACTIONS OF HUMAN TERM PLACENTA

The method is described in the text. The results of a representative experiment are presented. The values in parentheses are ranges of 3 to 6 separate experiments.

Fractions Specific activity (mlzmoles/io rain Ratio per mg protein) A[P

5-pregnene- 5-androstene- 3,2o-dione (P) 3,z7-dione (A)

Acetone powder method Crude extract 1.3 (o.8-I.7) 2.8 (2.3-3.2) 2.1 i. 5 M (NHa)2SO ~ 2.2 (o.8-3.o) 4.5 (3 .I-6.°) 2.0 3.0 M (NHa)2SO 4 I.O (o.6 2.0) 2. 4 (1.7-3.6) 2. 4

Deoxycholate method Crude extract 4.5 (3.5-5.6) 3.4 (3.1-4-3) 0.8 1. 5 M (NH4)2SO 4 2.9 (1.4-4.o) 2.6 (1.8-4.2) 0.9 3.0 M (NH4)2SO a 0.9 (O.6-1.2) I . I (1.o-1.4) 1.2

Biochim. Biophys. Acta, 122 (1966) 151-153

I52 SHORT COMMUNICATIONS

genate was stirred for 2 h at o-2 °, centrifuged at 2000 x g for IO min and subse- quen t ly at 78 ooo × g for I h. The superna tan t solution was fract ionated with am- mon ium sulfate as described above.

The assay systems for the 3-fl-hydroxysteroid oxidoreductase and 3-oxosteroid /14 ZlS-isomerase activities have been previously described 1. The 3-fi-hydroxysteroid oxidoreductase activities were determined with 5fi-androstan-3fl-ol-i7-one as sub- strate. The 3-fl-hydroxysteroid oxidoreductase ac t iv i ty was not observed with any of the fractions obtained.

The specific activities of the 3-oxosteroid ~4 AS_isomerase of the ammon ium sulfate fractions of human term placenta are shown in Table I. The ratios of the iso- merase activities with 5-pregnene-3,2o-dione (P) and 5-androstene-3,I7-dione (A) as substrates varied with the method of extract ion of the enzyme. The A/P ratios of the fractions obtained by the acetone powder method ranged from 2.0 to 2.4 and that of the deoxycholate method ranged from 0.8 to 1.2. These ratios differed from those of the subcellular fractions obtained by centr ifugation in a sucrose medium which ranged from 4-4 to 6.51. I t appears tha t t r ea tment with acetone or deoxycholate results in the solubilization of isomerase(s) with different activities for the two sub- strates. The activities for those two substrates were not separated by subsequent ammon ium sulfate fractionation. Furthermore, there was no increase in the specific activities. EWALD, WERBIN AND CHAIKOFF 2 reported, however, tha t the two activities were part ial ly separated by a m m o n i u m sulfate fract ionation of homogenates of bovine adrenal glands.

The undissolved sediment of the a m m o n i u m sulfate fractions were also assayed for activities. The results of one of the representat ive experiments are shown in Table II. Variat ion in the A/P ratios of the fractions obtained from acetone powder were noted. The ratios of the fractions obtained from the deoxycholate t r ea tment were consistent and lower than the superna tan t fraction (Table I). These variat ions in the A/P ratios in different fractions are consistent with the hypothesis tha t the isomerase for these two substrates are distinct.

TABLE Ii

3 _ O X O S T E R O I D / ] 4 / J S _ I S O M E R A S E A C T I V I T I E S IN T H E U N D I S S O L V E D S E D I M E N T OF T H E A M M O N I U M

S U L F A T E F R A C T I O N OF H U M A N T E R M P L A C E N T A

The method is described in the text.

Fractions Specific activities Ratio (mlimoles/zo rain per ~4 /P mg protein)

5-pregnene- 5-andro- 3,2o-dione stene-3,I 7- (P) dione (A )

Acetone powder method I. 5 M (NH4)2SO 4 0.8 1.8 2.2 3.0 M (NH4)2SOt 8 .6 I I . I 1. 3

Deoxycholate method 1. 5 M (NH,)2SO 4 5.1 3.2 0.6 3.0 M (NH~)2804 7.3 4-3 0.6

Biochim. Biophys. Acta, 122 (1966) 151-153

SHORT COMMUNICATIONS 153

OLEINICK AND KORITZ s reported tha t the addition of pyridine nucleotide to the assay system resulted in the stimulation of the 3-ketosteroid d4-AS-isomerase obtained from small particles of rat adrenal glands. The addition of 0. 5 #mole of NAD to the assay system of the placental isomerase from the ammonium sulfate fractions did not result in ally stimulation of the activity. I t is possible that the fractions did contain a small amount of pyridine nucleotides and that the addition of NAD did not increase the activity. The stimulatory influence of pyridine nucleo- tides might be dependent upon an intact particulate enzyme which is lost upon solubilization of the enzyme.

In this s tudy the 3-oxosteroid A4-AS-isomerase of human term placenta was isolated which was devoid of 3-/5-hydroxysteroid oxidoreductase activity. The re- sults suggest that the isomerase for 5-androstene-3,I7-dione and 5-pregnene-3,2o- dione are separate systems.

Laboratory of the Population Council, Rockefeller University, New York, N.Y. (U.S.A.)

SAMUEL S. KOIDE

MARIA T. TORRES

I S. S. KOIDE ANn M. T. TORRES, Biochim. Biophys. Acta, lO 5 (1965) 115. 2 W. EWALD, H. WERBIN AND I. L. CHAIKOFF, Steroids, 4 (1964) 759. 3 N. L. OLEINICK AND S. g . KORITZ, Biochemistry, 5 (1966) 715.

Received March 3oth, 1966

Biochim. Biophys. Acta, 122 (1966) 151--153

BBA 63189 Isolation of pure m-amylase from human saliva

The isolation and crystallization of salivary a-amylase (a-I,4-glucan 4-glucano- hydrolase, EC 3.2.1.1) by conventional precipitation methods with ethanol, acetone and ammonium sulphate has been reported by Muvs I and modified by FISCHER AND STEIN 2. While employing column chromatography on Sephadex G-25 and on calcium phosphate, MILLIN AND SMITH a eluted three active, heterogeneous fractions differing in stability. As yet, the homogeneity of purified salivary a-amylase has not been established.

The present paper describes a simple procedure for the isolation of pure, homo- geneous, a-amylase from human saliva. All operations have been carried out at room temperature. Amylase activity was determined by a modification of the method of BERN~ELD 4 for the assay of fl-amylase with dinitrosalicylic acid reagent, performing the enzymic digestions at 37 ° instead of 20 °. One amylase unit (AU) was defined as the amount of reducing sugar, liberated after 3 min reaction at pH 6. 9 at 37 °, which is equivalent to I mg of maltose. Protein content was estimated by the extinction at 28o m# (E 28o). A protein solution giving ~1 cm --~8o ms I.OO was defined as possessing one extinction unit (EU) per ml.

Biochim. Biophys. Acta, 122 (1966) 153-156