THE JO~NAL. 01 Browo~ca~ C~rsrmrav Vol. 252, No. 16. Issue of August 25. pp. 6904-5910, 1977

Prrnted ,n U.S.A.

Ribonuclease Inhibitor from Human Placenta PURIFICATION AND PROPERTIES*

PETER BLACKBURN, GLYNN WILSON, AND STANFORD MOORE

F&n The Rockefeller University, New York, New York 10021

(Received for publication, March 21, 1977)

A soluble ribonuclease inhibitor from the human placenta has been purified 4000-fold by a combination of ion ex- change and affinity chromatography. The inhibitor has been isolated in 45% yield (about 2 mg/placenta) as a protein that is homogeneous by sodium dodecyl sulfate-gel electrophore- sis. In common with the inhibitors of pancreatic ribonucle- ase from other tissues that have been studied earlier, the placental inhibitor is an acidic protein of molecular weight near 50,000; it forms a 1:l complex with bovine pancreatic RNase A and is a noncompetitive inhibitor of the pancreatic enzyme, with a Ki of 3 x 10-l” M. The amino acid composi- tion of the protein has been determined. The protein con- tains 30 half-cystine plus cysteine residues determined as cysteir acid after performic acid oxidation, At pH 8.6 the nondenatured protein alkylated with iodoacetic acid in the presence of free thiol has 8 free sulfhydryl groups. The inhibitor is irreversibly inactivated by sulfhydryl reagents and also by removal of free thiol from solutions of the protein. Inactivation by sulfhydryl reagents causes the dis- sociation of the RNase + inhibitor complex into active RNase and inactive inhibitor.

An inhibitor of neutral ribonuclease of the pancreatic type has been observed in the high speed supernatant fraction of a number of mammalian tissues (1). Nonmammalian tissues also contain this type of activity (2,3). The ratio of inhibitor to neutral RNase activity is high in those tissues characterized by high rates of RNA synthesis and RNA accumulation (4-6). The increase in RNA accumulation during regeneration of the rat liver following partial hepatectomy and in phytohemag- glutinin-transformed lymphocytes is preceded by a rise in the concentration of the RNase inhibitor (5, 7). Conversely, those tissues in which protein synthesis decreases and catabolic activity increases demonstrate lower levels of the inhibitor and elevated RNase activity (2, 8, 9).

The response of the inhibitor and neutral RNase to hor- monal and other metabolic stimuli has been investigated in a number of tissues. Following hypophysectomy of rats the liver inhibitor concentration was decreased; the inhibitor concen-

* This work was supported in part by United States Public Health Service, National Institutes of Health, Grant GM 07256 and a grant for research in reproductive biology from The Rockefeller Founda- tion. Preliminary reports on this research were presented at the meetings of the American Society of Biological Chemists (P. Black- burn and G. Wilson, (1976) Fed. Proc. 35, 1498; P. Blackburn and G. Wilson (1977) Fed. Proc. 36, 908).

trations increased on parenteral administration of growth hor- mone to the hypophysectomized animals (6). Liu et al. (10) have found an increase in the concentration of rat mammary gland inhibitor throughout gestation and particularly during the lactating period. These changes correlated with the accu- mulation of cellular RNA and increased protein synthesis. Steroid hormones have also been reported to affect the amounts of inhibitor and neutral RNase of mammalian tissues (11, 12); Zan-Kowalczewska and Roth (13) suggested identity for the RNase inhibitor and the estradiol receptor.

The integrity of polyribosomes during extraction from a tissue is greatly improved by the presence or addition of RNase inhibitor to inhibit endogenous RNase (14, 15). Re- cently, it has been demonstrated that the polyribosomal frac- tion isolated from mouse ascites cells contains a neutral RNase inhibitor; removal of this inhibitor from the ribosome by high ionic strength buffer decreased the stability of globin messen- ger RNA in an in vitro translation system (16).

The use of RNase and its derivatives to retard cell prolifera- tion in vitro and in uiuo (17-20) and the possible importance of the ribonuclease inhibitor in RNA metabolism and cellular development has prompted us to study the interaction between RNase and its endogenous inhibitor. Although many proper- ties of the partially purified inhibitor have been reported (21- 24), the structure of the protein and the nature of its interac- tion with RNase are still unknown. In part this reflects diffl- culties encountered in the extraction and purification of the highly unstable inhibitor present in small amounts in mam- malian tissues.

The RNase inhibitor of the human placenta (25, 26) was chosen for study. The placenta is a tissue of considerable biosynthetic activity, and it contains appreciable amounts of the inhibitor at full term. This paper is concerned with the preparation of inhibitor from this tissue.

EXPERIMENTAL PROCEDURES

Materials - Bovine pancreatic RNase A (type RAF) was purchased from Worthington. DTT,’ glutathione, 5,5’-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, p-hydroxymercuribenzoate, iodoacetic acid, l-chloro-3-tosylamido-7-amino-L.-2-heptanone HCl, L-l-tosyl- amide-2-phenylethylchloromethyl ketone, benzamidine hydrochlo- ride, cyclic 3’:5’-AMP and -GMP, and unlabeled steroid hormones were all obtained from Sigma. Yeast RNA (Sigma type VI, from Torula yeast) was dissolved in 0.025 M EDTA to give a 6% solution. The pH was adjusted to 6.5 to 7.0 with NaOH, and the solution was

’ The abbreviation used is: DTl’, dithiothreitol (Cleland’s re- agent).

5904

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Ribonuclease Inhibitor from Human Placenta 5905

dialyzed twice for 6 h against 20 volumes of 0.025 M EDTA, twice against 0.15 M NaCl, and three times against glass-distilled water and then lyophilized. Radioactively labeled steroids [2,4,6,7- 3Hlestradiol (91.3 Ci/mmol), 11,2,6,7-3Hlprogesterone (103.7 Ci/ mmol), I 1,2,4-3Hldexamethasone (22.6 Ci/mmol), and [1,2,6,7- 3H]cortisol (80 Cilmmol) were obtained from New England Nuclear. Sepharose 4B was purchased from Pharmacia Fine Chemicals. NJV- Dimethylformamide was from Eastman.

Assay for RNase and Inhibitor-RNase activity towards yeast RNA was determined by a modification of the precipitation method of Anfinsen et al. (27). The inhibitor of RNase was assayed for by a modification of the procedures of Roth (1) and Shortman (21). The incubation mixture contained 0.2 ml of 0.5 M Tris/HCl (pH 7.5), 5 rnM in EDTA, 0.1 ml of 0.1% bovine serum albumin, and 50 ~1 of the sample to be assayed. When inhibitor was to be assayed, 10 ng of RNase A in 10 ~1 of 0.1% bovine serum albumin were included in the assay mixture. When latent RNase (i.e. RNase complexed to inhibi- tor) was to be measured, 0.1 ml of 10 mMp-hydroxymercuribenzoate dissolved in 10 mM NaOH was included. In each case the volume was made up to 0.5 ml with glass-distilled water and the mixture was preincubated for 5 to 10 min at 37”. To start the reaction, 0.5 ml of 2% (w/v) yeast RNA, freshly prepared in glass-distilled water, was added and the mixture was shaken briefly on a Vortex mixer. Incu- bation was for 30 min at 37”. The reaction was terminated by the addition of 1.0 ml of ice-cold 10% (v/v) perchloric acid, 0.25% in uranyl acetate. The tubes were gently shaken and placed on ice for 30 min. The precipitates were centrifuged in a clinical centrifuge at 3000 x g for 20 min at 4’. A 0.5.ml aliquot was withdrawn from the supernatant and diluted lo-fold with glass-distilled water. The ab- sorbance at 260 nm was measured on a Zeiss PMQII spectrophotome- tar. Blanks, with and without sample, were also assayed.

Units of inhibitor activity were determined from a standard curve of percentage of inhibition (i,,) versus inhibitor units (& = 100[1/(K, + 1)J) (28). I equals units of inhibitor, where 1 unit is that amount of inhibitor required to inhibit the activity of 5 ng of RNase A by 50%. Hence, by definition 1 unit of inhibitor equals K,. The shape of the curve determined experimentally followed that for the theoretical binding curve obtained for the reversible formation of a 1:l complex. Inhibitor units were determined on solutions diluted to give approxi- mately 50% inhibition of the 10 ng of RNase A in the assay, since this represents the most accurate region of the standard curve.

This assay procedure was reproducible by a number of operators. The assay for RNase is linear in the range of 0 to 20 ng of RNase A with a final concentration of 1% yeast RNA in the incubation mix- ture. Routinely, 10 ng of RNase A gave an increase in absorbance at 260 nm of 0.4 under the assay conditions described. The linearity of the assay could be increased to above 25 ng of RNase A with the use of high molecular weight wheat germ RNA (Calbiochem) at a final concentration of 0.5% in the assay; this substrate was not routinely used.

It has been our experience that precipitation assay procedures used to determine RNase activity can be subject to considerable variation depending on the mode of addition of the precipitant, particularly if the volume of precipitant added is larger than the total volume of the incubation mixture. The assay method described here was not subject to this type of variation.

Preparation of SepharoselRNase A f29, 301- Sepharose 4B was washed with 20 volumes of glass-distilled water on a vacuum filter. The Sepharose was suspended in an equal volume of glass-distilled water, the pH was adjusted to 11, and the temperature was brought to below 10”. Cyanogen bromide (100 mglml of settled bed) was dissolved in a minimal volume of NJ-dimethylformamide (approxi- mately 1:l (w/v)), and the solution was added dropwise to the stirred Sepharose suspension. The pH was maintained at 11 by the addition of 4 M NaOH, and the temperature was maintained below 10”.

The rate of activation reaction may be controlled easily by the rate of addition of the CNBr solution. The activation of 50 ml of Sepharose 4B is complete after approximately 30 min, as judged by the end of NaOH uptake required to maintain pH 11.

The activated Sepharose was washed with 10 volumes of ice-cold 0.1 M NaHCO,, pH 9.5, on a vacuum filter. To 40 ml of CNBr- activated Sepharose, suspended in 40 ml of NaHC03 (pH 9.5), were added to 50 mg of RNase A. The suspension was gently stirred for 20 h at 4”. The suspension was vacuum filtered and then resuspended in 0.1 M TrislHCl, pH 7.5, for 4 h at 4”. The Sepharose/RNase was then washed with successive 20-volume aliquots of 0.1 M NaHCO,, 2 M NaCl, and finally distilled water. The Sepharose/RNase was stored in glass-distilled water at 4”.

The amount of RNase A eluted in the washes was determined with cyclic 2’,3’-cytidylic acid as substrate (31). The amount of RNase A coupled to the Sepharose was determined directly by the ninhydrin reaction after alkaline hydrolysis (32, 33) of a lyophilized sample. Activated Sepharose was used as a control. Both methods indicated that approximately 1 mg of RNase A was coupled per ml of settled Sepharose.

The functional capacity of the affinity adsorbent was determined by saturating a small column of the SepharoselRNase with an excess of inhibitor at pH 6.4 in phosphate buffer. The units of inhibitor eluted at pH 5.0 by 3.0 M NaCl were determined. Approximately 40,000 units of inhibitor were recovered per ml of settled bed.

Stability of the SepharoselRNase - A small column containing 2 ml of settled bed was washed with successive lo-volume aliquots of 20 rnM TrislHCl (pH 7.5), 2 M in NaCl, or 20 rnM phosphate (pH 7.0), 2 M in NaCl. A small amount of RNase was obtained in a Tris buffer wash in contrast to negligible enzyme activity in a phosphate eluent. Repeated use and washing of this affinity gel over a 3-month period with both 45 mM potassium phosphate (pH 6.4) and 20 mM acetate (pH 5.0), 3 M NaCl, did not lead to any detectable enzyme activity in the eluents. This deleterious effect of Tris and other amino-contain- ing buffers on the stability of ligands attached to CNBr-activated Sepharose was first reported by Wilchek et al. (34).

Amino Acid Analysis - Hydrolyses were routinely carried out with 6 N HCl in glass tubes sealed under vacuum (50 pm Hg) at 110”. The analyses were performed on a Durrum D-500 automatic amino acid analyzer.

Performic acid oxidation of samples was carried out according to the procedure of Moore (35). Prior to hydrolysis, evaporation of the sample under reduced pressure was repeated in the 90% formic acid to ensure complete removal of the HBr used to quench the performic acid.

Carboxymethylation of protein samples was carried out with io- doacetic acid for 2 h at room temperature in 0.1 M Tris buffer, pH 8.6. A 2-fold molar excess of the reagent over total thiol concentration was used. Carboxymethylcysteine was determined on the amino acid analyzer after acid hydrolysis of the protein, which had been de- salted by dialysis in the dark against 0.1 M acetic acid.

Steroid Binding Assays - The method of Ginsburg et al, (36) was used to distinguish high affinity binding from nonspecific binding. Columns (12 x 0.5 cm) of Sephadex G-25 equilibrated in 20 rnM Trisl HCl (pH 7.5), 5 mM in DTT, and eluted at O-4”, were used to separate protein from labeled steroid. Fractions of 200 ~1 were collected, and 170.kl aliquots were added to 10 ml xylene-based scintillation fluid (Aquasol Universal LSC, New England Nuclear). Tritium was counted at an efficiency of 25%. Solutions containing 150 ~1 of inhibitor solution (2000 units/ml) and either 20 ~1 of 20 rnM Tris buffer (pH 7.5), 5 rn~ in DTT, or 20 ~1 of this buffer, lo-” M in unlabeled steroid, were incubated at 25” for 30 min in the presence of 20 ~1 of 2 to 3. x 10 R M 3H-labeled steroid. Controls with buffer in place of inhibitor solution were also incubated. Diethylstilbestrol was used as the unlabeled competitor to 17P-estradiol (36).

Measurement of DTT- The concentration of DTT in placental homogenates was determined by 5,5’-dithiobis(2-nitrobenzoic acid) (37). The reagent solution was calibrated against a freshly prepared solution of DTlY (range, 0 to 100 nmol of -SH/ml). After homogeni- zation, placental extracts were stored in tightly stoppered glass vials and were assayed immediately after being opened.

Gel Electrophoresis - Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was performed by the method of Weber and Osborn (38). Isoelectric focusing was performed on the inhibitor (obtained after Step 5) in 7.5% acrylamide gels (85 x 5.5 mm), 12% in sucrose and 1% in Ampholine (pH range, 3 to 10; LKB). After electrophoresis (0.5 mA/gel, 16 h), gels containing protein were fixed for 2 h in 10% trichloroacetic acid and then were stained with Coomassie blue (62.5 mg in 454 ml of methanol and 64 ml of glacial acetic acid). Bovine serum albumin (the isoelectric point, p1, was 4.8) was tested as a standard.

Protein Estimation - During the purification procedure protein was determined according to Lowry et al. (39), with bovine serum albumin as a standard, or spectrophotometrically at 280 nm.

RESULTS

Purification of RNase Inhibitor

All operations were performed at O-4”. All buffers were 5 mM in D’lT and 1 mM in EDTA.

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5906 Ribonuclease Inhibitor from Human Placenta

Step 1: Preparation of Homogenate - Human placentas ob- tained within 30 min of normal term delivery were stripped of membranes and washed with 20 mM Tris/HCl (pH 7.5),0.25 M in sucrose. The tissue was cut into segments, blotted on filter paper to remove excess blood, and homogenized in the same buffer 1:2 (w/v) for 90 s at Setting 10 in a Sorvall Omni-Mixer maintained in an ice bath.

The homogenate was centrifuged at 16,000 x g for 30 min, and the supernatant was retained. The supernatant contained 30 to 40 units of inhibitor/mg of protein (approximately 10 pg of inhibitor/g of tissue, wet weight). The amount of free RNase activity was approximately 0.045 pg of RNase A equivalents;

20

ii- 20 40 60 80

Fraction number

100

FIG. 1. DEAE-cellulose column chromatography of the 35 to 50% ammonium sulfate fraction. The sample was applied to the column (30 x 2.6 cm) in 20 mM Tris/HCl (pH 7.5), 1 mM in EDTA, 5 mM in DTT. The column was then washed with the Tris buffer containing 0.15 M NaCl (the breakthrough protein peak is not shown in the figure). The inhibitor was eluted with a linear gradient (300 ml in each chamber) of NaCl from 0.15 to 0.5 M in the same Tris buffer, pH 7.5, and 5.0-ml fractions were collected. 60, absorbance at 280 nm; - - -, NaCl gradient; O---O. inhibitor activity as percentage of inhibition of 10 ng of RNase A.

f 100 L

6 .- 5

80 r .c ,\”

the amount of latent RNase activity was approximately 0.461 pg of RNase A equivalents per g of t&sue, wet weight.

Steps 2 and 3: Ammonium Sulfate Fractionation - Solid ammonium sulfate was added slowly to the stirred superna- tant from Step 1 to achieve 35% saturation. The suspension was gently stirred for 60 min and then was centrifuged at 16,000 x g for 30 min. The supernatant solution was removed and brought to 50% saturation with ammonium sulfate. After 60 min, the suspension was centrifuged as before. At this stage the 35 to 50% ammonium sulfate precipitate could be stored, either overnight at 4” or at -15” over a period of weeks, with no significant loss of activity. The 35 to 50% ammonium sul- fate precipitate was dissolved in 50 to 100 ml of Tris/HCl buffer, pH 7.5, and then dialyzed twice for 4 h against 20 volumes of the same buffer. The dialyzed fraction was then centrifuged at 48,000 x g for 1 h.

Step 4: DEAE-cellulose Chromatography- The superna- tant solution from Step 3 was applied to a column (30 x 2.6 cm) of DEAE-cellulose (Whatman DE52) equilibrated with 20 mM Tris/HCl buffer, pH 7.5. The column was washed with this buffer containing 0.15 M NaCl until the breakthrough peak of protein had been eluted. This peak contained placental RNase complexed to inhibitor and approximately 90% of the total protein. The column was then eluted with a 600-ml linear gradient: 0.15 to 0.5 M NaCl in Tris/HCl buffer, pH 7.5. A typical elution profile is shown in Fig. 1. The peak of RNase inhibitor activity was eluted at about 0.24 M NaCl concentra- tion.

Step 5: Affinity Chromatography (Fig. 2) -The pooled frac- tions containing inhibitor from Step 4 were dialyzed against two changes, each of 20 volumes of 45 mM potassium phos- phate buffer, pH 6.4, for 6 h. The dialyzed fraction was applied to a column of Sepharose/RNase (volume of settled bed, 8 ml) equilibrated in 45 mM potassium phosphate buffer, pH 6.4. After the sample application, the affinity column was washed with 0.5 M NaCl in the pH 6.4 buffer. This wash removed a small amount of noninhibitor protein from the column. The nature of this protein has not been determined, but probably it simply reflects protein nonspecifically bound to the basic

- 28,600

FIG. 2 (left). Affinity chromatogra- phy of inhibitor fraction from Step 4 dialyzed against 45 mM potassium phosphate buffer, pH 6.4. At A the eluent was changed to pH 6.4 buffer, 0.5 M in NaCl. At B elution was initi- ated with 20 mM acetate buffer (pH 5.0), 3.0 M in NaCl and 15% (v/v) in glycerol. Fractions of 1.0 ml were col- lected.

FIG. 3 (right). Polyacrylamide gel electrophoresis in sodium dodecyl sul- fate of pooled fractions after Step 4 of the purification procedure (a); purified inhibitor after Step 5 (b); and molecu- lar weight markers (c) (BDH Chemi- cals Ltd., England).

IO 20 30 40

Fraction number

a b c

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Ribonuclease Inhibitor from Human Placenta 5907

RNase A molecule. The inhibitor was eluted with 20 mM acetate (pH LO), 3.0 M in NaCl and 15% (v/v) in glycerol. The preparation obtained at this stage gave a single band on gel electrophoresis in the presence of sodium dodecyl sulfate, as shown in Fig. 3, and upon isoelectric focusing (p1 = 4.6 to 4.7).

The purification procedure is summarized in Table I. The specific activity of the pure inhibitor was 100,000 unitslmg of protein, corresponding to a 4,000-fold purification over the initial extract, with an overall recovery of approximately 45%. In the final stages of purification, the presence of 15% (v/v) glycerol or 0.1% bovine serum albumin in the buffer for elution is essential for good recovery of inhibitor activity.

The recoveries of inhibitor indicated in Table I depend upon the use of buffers 5 mM in D’IT. When 1 mM D!lT was used at the start of this work, there was a greater than 50% loss of inhibitor activity in the initial supernatant within 24 h. Sig- nificant loss of activity was also observed during ammonium sulfate fractionation. In solutions 1 mM in DlT, the stability of the inhibitor in initial extracts was not improved by centrifu- gation of the homogenate at 100,000 x g for 1 h or by the use of buffers containing the protease inhibitors, 1-chloro-3-tosylam- ido-7-amino-L-2-heptanone HCl (1 mM), Ll-tosylamide-2- phenylethylchloromethyl ketone (0.1 mM), or benzamidine hy- drochloride (1 mM).

The loss of inhibitor activity was related to the loss of DTT from the initial extract. When the extracting buffer was 5 mM in DlY’, determination of the DTl’ concentration of the homog- enate immediately after extraction showed the concentration to be 2 to 2.5 mM. The concentration of D!lT in initial extracts became progressively lower with storage. As shown in Table II, there was a marked loss of inhibitor activity when the DTT concentration approached 1 mM.

The DIT concentration in samples of pure inhibitor was adjusted by dialysis for 20 h at 4” against 20 mM acetate (pH LO), 1 rnhf in EDTA, 15% (v/v) in glycerol, 0.15 M in NaCl, and 0.1 to 5.0 mM in DTT. Inhibitor was obtained fully active in either 1 or 5 mM DTT immediately after dialysis. After 1 week of storage at 4”, the sample that was 1 mM in DlT had lost about 60% of its activity. Samples dialyzed against either 0.1 or 0.5 mM DTT showed significant loss of activity. Inhibitor activity was not regained upon addition of DTT to 5 mM in these solutions.

The inhibitor was fully stable for over 2 months when stored at 4” or at -15” in the buffer used to elute the protein from Sepharose/RNase.2 Rapid refreezing and thawing of the pure inhibitor solutions resulted in some loss of activity (20 to 30% loss after freezing and thawing two times, 60% loss after freezing and thawing 20 times).

Removal of either glycerol or bovine serum albumin from pure inhibitor solutions resulted in almost complete loss of activity. Significant loss of activity was also found during concentration on Diaflo ultrafiltration membranes (LJM-10 and PM-lo. Amicon Corp.).

Molecular Weight

The molecular weight of the pure inhibitor estimated by gel filtration on Sephadex G-100 in 20 mM acetate (pH 5.0), 1 mM in EDTA, 5 mM in DTT, 15% (v/v) in glycerol, 0.15 M in NaCl, for three different preparations was about 52,000 (Fig. 4). The molecular weight estimated by sodium dodecyl sulfate-gel electrophoresis was 50,000. The molecular weight of the com-

r The inhibitor is also stable when dialyzed into 20 mM TrislHCl (pH 7.5). 1 mM in EDTA, 5 mM in DTT, 0.15 M in NaCl, 15% (v/v) in glycerol, and stored frozen at - 15”.

TABLE I

Purification of RNase inhibitor from human term vlacenta

SbP Total Total in- Specific RMOV-

motein hibitor activitv erv

w? units unitslmg 96 1. Initial extract 16,000 400,000 25 100 2. 35 to 50% ammonium 2,327 320,000 137.5 80

sulfate precipitate 3. 46,000 x 1 h g, 1,646 320,000 194 80 4. DEAE-cellulose chro- 308 240,000 780 60

matography 5. SepharoselRNase A 1.6 180,000 100,000 45

affinity chromatog- raphy

TABLE II

Relationship between the concentration ofDTT and the actiuity of placental RNase inhibitor in initial extracts

Extt;:fng Time after homog- DTT concentra- Inhibitor a enization tion activiW

Tris

Phosphate

h m&f %

2 2.3 100 24 1.7 63 46 0.5 13

2 3.1 100 24 2.7 100 48 1.2 66 72 1.0 27

a Extracts of placental tissue were made in either 20 rnM Tris buffer, pH 7.5, or45 rnM phosphate buffer, pH 6.4; both buffers were 1 mM in EDTA, 5 rnM in DTT, and 0.25 M in sucrose.

* Inhibitor activity measured 2 h after preparation of the initial extract was taken as 100%.

i I 1 I , 1 42 46 50

Log molecular weight

FIG. 4. Estimation of the molecular weight of the human placen- tal RNase inhibitor by gel filtration on a Sephadex G-100 column (1.5 x 90 cm). The column was eluted with 20 mM acetate buffer, pH 5.0, containing 1 mM EDTA, 5 IIIM D’ll’, 0.15 M NaCl, 15% (v/v) glycerol at 4”. The column was calibrated with the protein standards: 1, RNase A; 2, horse myoglobin; 3, trypsin; 4, cu-chymotrypsin; 5, ovalbumin, and 6, bovine serum albumin. The samples were applied in 1.0 ml of buffer, and 1.5-ml fractions were collected. V,, the elution volume of each protein; void volume (VJ was determined with blue dextran.

plex of inhibitor with RNase A (M, 13,700) was 64,000 esti- mated by gel filtration on Sephadex G-100. The molecular weight of the complex is consistent with a stoichiometry of 1:l for the binding of inhibitor to RNase A.

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5908 Ribonucleuse Inhibitor from Human Placenta

pH Optimum

The pH profile for the inhibition of RNase A by the human placental inhibitor shows maximal activity between pH 7 and 8, with the optimum at pH 7.2 to 7.5.

Kinetics of Inhibition and the Effect of Sulfhydryl Reagents

The inhibition of RNase A by the inhibitor in 0.1 mM Tris, 1 mM in EDTA, pH 7.5, is noncompetitive and has a Ki for the inhibitor of about 3 x lO-*O M as demonstrated by the Dixon plot, l/V against [Zl (Fig. 5), and the double reciprocal plot, l/ V against l/S -(Fig. 6). When the buffer is 1 mM in p-hydroxy- mercuribenzoate, the inhibitor is completely inactive. This inactivated species of the inhibitor has no effect on the kinetics of RNase A action toward yeast RNA (Fig. 6).

The inactivation of the inhibitor withp-hydroxymercuriben- zoate has been found to be irreversible; dialysis of such a sample against a lOOO-fold excess of DTT at pH 7.5 failed to reactivate the inhibitor.

The inactivation of the inhibitor by either 1 mM p-hydroxy- mercuribenzoate or N-ethylmaleimide brings about the disso- ciation of the inhibitor. RNase complex. Gel filtration of the p- hydroxymercuribenzoate-inactivated complex with an eluting buffer 1 mM in p-hydroxymercuribenzoate or of the N-ethyl- maleimide inactivated complex led to recovery of all the RNase activity as a M, = 13,700 species.

Effect of Steroid Hormones, Cyclic Nucleotides, and Divalent Metals

None of the steroid hormones, progesterone, 17@-estradiol, dexamethasone, or cortisol, bound specifically to the purified inhibitor under the conditions of the radioactive assay. In some experiments trace amounts of radioactivity obtained in the protein peak from Sephadex G-25 chromatography were also found in experiments that contained a 50-fold molar ex- cess of unlabeled steroid, suggesting a nonspecific association of steroid with inhibitor. When included in the normal assay for RNase inhibitor, none of the above steroid hormones or the cyclic nucleotides, 3’,5’-AMP and 3’,5’-GMP, in concentra- tions of 1 mM effected the reversal of inhibition of RNase A by the inhibitor. The divalent cations, Ca2+, Mg%+, or Zn2+ (at 0.1 to 10 mM), similarly had no effect.

J

[InhIbItor] M x IO”

40 -30 -20 -10 IO 20 30 40

;

FIG. 6 (right). The kinetics of inhibition of RNase A by the hu- FIG. 5 (left). Kinetics of inhibition of bovine pancreatic RNase A by the human placental ribonuclease inhibitor. The data are pre- man placental inhibitor and the effect ofp-hydroxymercuribenzoate. sented in a Dixon plot, l/V against [I], at a saturating substrate The data are presented in a double reciprocal plot, l/V against l/S. concentration of 1% yeast RNA. The activity determinations were Activity determinations on 10 ng of RNase A were made in the carried out as described under “Experimental Procedures” at pH 7.5 presence OE X, 13.3; 0, 26.6; l , 36.9; q ,53.2 ng of pure inhibitor; 8,O for two different enzyme concentrations; 0, 10 ng of RNase A; A, 15 ng of inhibitor or 53.2 ng of inhibitor in the presence of 1 rnM p- ng of RNase A. Under these conditions the assay for RNase A is hydroxymercuribenzoate. All other assay conditions were as de- linear up to 20 ng of RNase A. The lines drawn through the points scribed under “Experimental Procedures.” were calculated from a least squares tit to the data.

Amino Acid Composition

The amino acid composition of the human placental inhibi- tor is shown in Table III. Total l/z cystine + cysteine was determined as cysteic acid atier performic acid oxidation of the protein, and it indicates 30 residues of l/z cystine + cysteine per molecule of inhibitor. The free sulfydryl content of the nondenatured protein (100 pg) was determined by carboxy- methylation with iodoacetic acid at pH 8.6 in 0.1 M Tris buffer, 1 mM in EDTA, 2.5 mM in D’IT, 15% (v/v) in glycerol (total volume, 1 ml). Alkylation was carried out in the dark for 2 h at

TABLE III

Amino ueid composition of human placental RNase inhibitor Amino acid Ftesidues/moleculea

Asx 47 Threonineb 16 Serine’ 45 Glx 64 Proline 17 Glycine 36 Alanine 34 Valine 24 Methionine 2-3 Isoleucine 12 Leucine 65 Tyrosine b 4 Phenylalanine 6 Histidine 6 Lysine 17 Arginine 23

‘/a Cystine + Cysteine” 30 Tryptophan’ -5 Total residues 473

u Mean analyses (average of values) of six separate preparations; the residue ratios were calculated for a molecular weight of 51,000.

@ Corrected for 5% loss during 20-h hydrolysis (40). c Corrected for 10% loss during 20-h hydrolysis (40). d Determined as cysteic acid after performic acid oxidation (35).

Carboxymethylation of the nondenatured protein at pH 8.6 yielded 6 carboxymethylcysteine residues.

p Tryptophan was determined following alkaline hydrolysis in the presence of thiodiglycol (41), scaled down to the use of 0.1 ml of 4 N NaOH.

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Ribonuclease Inhibitor from Human Placenta 5909

room temperature. Under these conditions the inhibitor has 8 free sulihydryl groups/molecule. No significant increase in the isoleucine and valine values was obtained after prolonged acid hydrolysis for 48 and 72 h, which suggests no Val-Val, Ile-Ile, or Ile-Val sequences in the molecule. No evidence for amino sugars was found in any of the analyses, nor was evidence for reducing sugars found by the phenolsulfuric acid assay (42).

DISCUSSION

The human placental neutral ribonuclease inhibitor is a protein similar to that reported for a number of mammalian tissues. It is present in the postmitochondrial supernatant of the placental extracts and is optimally active at physiological pH and ionic strength. Determinations of the free and latent neutral RNase activity measured at pH 7.5 in these extracts indicated that more than 90% of the total RNase activity is inactive, bound in a complex with its inhibitor.

The preparation of homogeneous RNase inhibitor in suffi- cient quantities to study the structure and function of the protein has been facilitated by the use of an efficient affinity chromatography system. The use of a solution of cyanogen bromide in dimethylformamide facilitates the activation of Sepharose 4B. The high capacity of the affinity column ena- bles milligram quantities of the homogeneous inhibitor pro- tein to be prepared with good recovery of activity, which is stable for months. Compared to the carboxymethyl cellulose/ RNase used by Gribnau et al. (43) and by Gagnon and de Lamirande (24), the SepharoselRNase has a functional capac- ity more than 10 times greater and the recovery of active inhibitor is higher. The protective effects of EDTA and thiols on the inhibitor protein have been reported previously (22). However, the data presented here show that in the prelimi- nary stages of purification free thiol, as DTT, is being depleted at an accelerated rate in the extracts and that this depletion can lead to the eventual loss of inhibitor activity. The critical concentration for loss of inhibitor activity appears to be about 1 mM DTl’. Loss of free thiol from preparations after DEAE- cellulose chromatography was not observed to exceed the nor- mal half-life for DTI’ in neutral buffer at 4”.

The inhibitor has a high binding affinity for RNase A, as demonstrated by the low K, and by the conditions required to elute the protein from the RNase A affinity column. The human placental inhibitor is inactivated by sulfhydryl block- ing reagents, similar to the RNase inhibitors reported from other sources (1). Inactivation of the inhibitor by p-hydroxy- mercuribenzoate yielded a species that no longer interacted with the RNase (Fig. 6). The kinetic data of RNase A action in the presence of the p-hydroxymercuribenzoate-treated inhibi- tor were identical with those for the RNase A alone. Since heparin and other polyanions are known to be competitive inhibitors of RNase activity (for review see Ref. l), it was possible that the acidic inhibitor protein might still interact with the RNase as a competitive inhibitor. No such interaction occurs between the inhibitor and RNase A.

The sullhydryl blocking reagents, p-hydroxymercuriben- zoate and N-ethylmaleimide, caused the complete dissociation of the complex into active RNase and inactive inhibitor, as shown by gel filtration. This finding is contrary to a result observed with the rat reticulocyte native inhibitor.RNase complex (44); although the inhibitor was inactivated by p- hydroxymercuribenzoate and generated active ribonuclease, the complex did not dissociate, but the pH and ionic strength for dissociation were different from ours.

A number of reports have suggested that the inactivation of the inhibitor by sulfhydryl reagents may be reversed by the addition of an excess of free thiol (23, 44, 45). In our experi- ments the inactivation of the human placental RNase inhibi- tor by p-hydroxymercuribenzoate or upon removal of DTT from storage buffers by dialysis has been irreversible. Experi- ments designed to reactivate the inhibitor by dialysis against 5 mM DlT were unsuccessful.

In consideration of the evidence supporting regulation of the neutral RNase and its inhibitor in response to hormonal stim- ulus, we have made a preliminary investigation into such interactions with the purified inhibitor. Since none of the steroid hormones tested bound specifically to the pure inhibi- tor, the observed hormonal effects (11, 12) on the tissue RNase and its inhibitor in vivo do not appear to depend upon their direct interaction with the RNase and its inhibitor. Murthy and McKenzie (46) suggested that the effect of thyrotropin on the rat thyroid inhibitor was mediated via cyclic nucleotides; we find no effect of cyclic nucleotides on the inhibitor interac- tion with RNase A.

The amino acid composition of the protein, as shown in Table III, demonstrates a large number of Asx and Glx resi- dues, a sufficient number of which must be aspartic acid and glutamic acid residues to give the protein ita acidic isoionic point. There are a large number of leucine residues; leucine represents the single most abundant residue. The most strik- ing feature is the large number of l/z cystine + cysteine residues determined as cysteic acid following performic acid oxidation of the protein. Carboxymethylation of the native protein at pH 8.6 in the presence of an excess of free thiol indicated 8 free sulthydryl groups/molecule. This result indi- cates that the ribonuclease inhibitor may have as many as 11 disulfides/molecule. The data on the irreversible inactivation of the inhibitor by sulthydryl reagents and by removal of DTT from storage buffers suggest that a highly organized confor- mation of the protein is required for its interaction with the RNase and that this conformation depends upon the -SH and S-S groups within the molecule.

The structure of the inhibitor protein and the precise role of its sulthydryl and other functional groups involved in the interaction between the inhibitor and the RNase are currently under investigation. The availability of milligram quantities of the RNase inhibitor in a pure and stable form, isolated by the purification procedure reported here, should be helpful in the control of endogenous RNase activity in in vitro transla- tion and transcription studies.

Acknowledgments -We acknowledge the counsel of Dr. William H. Stein in the course of this work and the skilled technical assistance of Paul Montalban. We thank Dr. F. Fuchs and the staff at the New York Hospital-Cornell Medical Center for making available human placentas. The typescript was prepared with the skillful assistance of Mrs. Frances S. Pearson.

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